WO2005085415A1 - Novel transformant and process for producing polyester by using the same - Google Patents

Abstract

It is intended to provide a yeast which has an excellent cell productivity, can be easily handled in gene manipulation and has auxotrophy achieved by exclusively disrupting specific genes; a method of constructing the transformant; and a process for producing a gene expression product, in particular, a polyhydroxyalkanoic acid. Namely, a yeast in which a plural number of genes have been disrupted is constructed by homologous recombination. Further, genes of enzymes participating in the polyhdyroxyalkanoic acid synthesis (for example, polyhydroxyalkanoate synthase gene and acetoacetyl CoA reductase gene) are transferred into this yeast with gene disruption to give a transformant. Furthermore, this transformant is cultured so as to efficiently accumulate a polyester copolymer comprising the polyhydroxyalkanoic acid in the cells followed by the collection of the polymer from the culture.

Description

 Specification

 New transformant and method for producing polyester using the same

 Technical field

 The present invention relates to a gene-disrupted strain in which a specific chromosomal DNA of yeast is disrupted by the principle of homologous recombination. It also relates to the production of industrially useful substances using the disrupted strain.

 Further, the present invention relates to a gene required for enzymatically synthesizing the copolyester, a microorganism for fermenting and synthesizing the polyester using the gene, and a method for producing a polyester using the microorganism. Furthermore, the present invention relates to a method for breeding the microorganism.

 Background art

 [0002] Advances in genetic recombination technology have made it possible to produce industrially useful substances in large quantities using prokaryotes and eukaryotes. Among eukaryotes, yeast, Saccharomyces, has long been used for the production of fermentable foods such as alcoholic beverages, and has been used as a microbial protein in Candida maltosa for a long time. The safety of this has been confirmed.

 [0003] Yeasts can be cultured at a higher cell density than bacteria, which generally grow faster. Furthermore, yeast is easier to separate bacterial cells and culture liquid than bacteria, so that the process of extracting and purifying the product can be simplified. By virtue of these properties, yeast has been used as a host for producing useful products from recombinant DNA, and its usefulness has been demonstrated.

 [0004] Among various yeasts, Candida yeast differs from Saccharomyces in that it does not produce ethanol in aerobic culture and does not inhibit growth due to it, so continuous culture at high density is possible. Thus, efficient cell production and substance production are possible. In addition, the spore-free yeast Candida maltosa has a carbon chain C

 6-C straight chain hydrocarbons, palm oil, coconut oil 40

It has the property of assimilating and growing fats and oils as the only carbon source! Since this property is practically advantageous as a place for producing useful substances or converting by converting hydrophobic substances, it is expected to be used for production of various compounds (see Non-Patent Document 1). . In addition, it is expected that Candida's maltosa can be used for production of useful substances by genetic recombination by virtue of its properties, and gene expression systems for this purpose have been vigorously developed (Patent Document 1, 2). Recently, it has been disclosed that Candida maltosa can be used for producing linear dicarboxylic acids by genetic recombination (see Patent Document 3) and biodegradable plastics (see Patent Document 4).

[0005] Thus, a host vector system for Candida maltosa was developed from an early stage, and despite the fact that many auxotrophic mutants were obtained by mutagenesis, There is no industrial drama of producing new useful chemical substances using the body. The reason for this is that no Candida maltosa with appropriate auxotrophy has been obtained that has the same growth ability as the wild type and the ability to assimilate linear hydrocarbon chains. The CHA1 strain developed by mutating Candida's maltosa has been confirmed to have mutations in the A DEI gene and the HIS5 gene (see Non-Patent Document 2), but its growth ability is inferior to that of the wild strain. . This is thought to be due to mutations other than the target.

[0006] When yeast is used as a host for substance production by genetic recombination, a selection marker (selection symbol) that can confirm that the target gene has been introduced is used, as in the case of using E. coli or the like. In the case of yeast, a gene that imparts resistance, such as cycloheximide ゃ G418 or hygromycin B, is used as a selectable marker gene that imparts drug resistance. However, there are known problems such as the fact that bacteria that do not carry the target gene may grow slightly because there is no sharp drug for yeast, and that the degree of drug resistance gradually increases. I have. Furthermore, the degree of drug resistance differs for each yeast strain, and the amount of expression of the drug resistance gene required for conferring drug resistance in yeast differs. Therefore, in order to impart drug resistance to each yeast, a promoter for expressing the drug resistance gene must be appropriately selected or prepared. In particular, Candida's maltosa has a strong initial resistance to cycloheximide (see Non-Patent Document 3), and the degree of resistance to various drugs as described above is not known. In addition, some yeast species, such as Candida maltosa, are known to codon translation in a manner that is different from the general format of E. coli and humans. Reference 4). That is, there is a high possibility that the drug resistance gene cannot be used directly.

[0007] Therefore, an appropriate auxotrophic selection marker is preferably used. In order to use an auxotrophic selection marker, it is necessary to obtain a mutant to which auxotrophy has been added. Conventionally, a mutant strain has been obtained from auxotrophic rice cake by random mutagenesis using a mutagen such as nitrosoguanidine or ethyl methanesulfonic acid. However, although the target auxotroph can be obtained by this mutagenesis method, it cannot be denied that the mutation may be present at a site other than the target site. As described above, this is an obstacle to development using yeast as a host, and it can be said that utilization as a place for producing substances is delayed compared to E. coli and the like.

 [0008] Another problem of the mutant strain obtained by random mutation is the natural return of the mutation site. In this case, since the revertant strain grows preferentially during the culturing, the productivity of the recombinant bacterium may decrease. In addition, the reverted strain has a problem in terms of safety standards that have a high possibility of surviving and multiplying when it flows out into the natural world. Therefore, it is not appropriate to use a strain obtained by random mutagenesis as a place for producing a substance. Therefore, it is desired to obtain a disrupted strain in which only genes involved in the synthesis of specific amino acids, vitamins, and the like have been disrupted, and AC16 has been added as an auxotrophic candy by disrupting only the ADE1 gene. A strain was produced (see Patent Document 5). However, this strain has a problem in that the gene that can be introduced is limited due to the single marker.

 [0009] Methods for introducing a gene into yeast include a method using a plasmid vector and a method for incorporating the gene into a chromosomal gene.

Plasmid vectors, which are genes capable of autonomous replication in yeast cells, are classified into two types: a type that has about one copy in one cell (YCp type) and a type that can have multiple copies (YRp type). Is being developed. Use of the latter plasmid vector Power that is considered to be advantageous for increasing the expression level of the target gene product In general, there are many problems with the stability of the plasmid vector, which is an industrial advantage In many cases, it cannot be used. In such a case, a promoter for expressing the target gene using YCp type It is considered to make one more powerful or to increase the number (copy number) of genes to be introduced. It is known that when a gene is introduced into a yeast using a plasmid vector, the size of the introduced gene is limited. Depending on the type of plasmid vector used, it is difficult to prepare a vector containing too large a gene, such as when multiple types of genes are to be introduced or when a single gene is to be introduced. It is hardly industrially advantageous in that the efficiency of introduction of the vector into yeast decreases, the target gene is deleted in yeast, and the like. In such a case, the problem can be solved by integrating the target gene into the chromosome. Also, the target gene may be more highly expressed when integrated into the chromosome. However, when there is only one type of selectable marker, once the selectable marker is used, the transgenic strain no longer has the selectable marker, and multiple gene transfer is impossible.

 Therefore, a gene-disrupted yeast having multiple auxotrophic markers is desired!

[0010] As described above, as mutants of Candida maltosa, gene mutations such as ADE1 gene, histidinol phosphate aminotransferase (HIS5 gene), orotidine 5, -phosphate decarboxylace (URA3 gene), etc. Many strains have been acquired (see Non-Patent Document 1). However, it was difficult to obtain Candida's maltosa to which multiple auxotrophy was added by specifically disrupting only a specific gene because the yeast showed a partial diploid. For this reason, in order to utilize the characteristics of yeast and to construct an industrially useful substance production system by gene recombination, it has been desired to develop a host having a plurality of selectable markers by gene disruption. Further, not only Candida maltosa but also such a gene-disrupted strain is desired.

 In addition, Candida maltosa, which has multiple selectable markers, produces industrially useful substances by utilizing the characteristics of assimilating and growing straight-chain hydrocarbons, palm oil, palm oil, and other fats and oils as the sole carbon source. Is expected.

[0011] In addition, it is known that many microorganisms accumulate polyesters such as polyhydroxyalkanoic acid (hereinafter abbreviated as PHA) as an energy storage substance in cells. A typical example is a homopolymer of 3-hydroxybutyric acid (hereinafter abbreviated as 3HB). A poly-3-hydroxybutyric acid (hereinafter abbreviated as P (3HB)), which was first discovered in 1925 in Bacillus megaterium (Non-Patent Document 5). P (3HB) is a thermoplastic polymer, which has been attracting attention as an environmentally friendly plastic because it is biodegradable in the natural environment. However, P (3HB) has high crystallinity and is hard and brittle, so its practical application is limited. For this reason, research has been conducted to improve this property.

 Among them, production of a copolymer (hereinafter, P (3HB-CO-3HV) t) composed of 3-hydroxybutyric acid (3HB) and 3-hydroxyvaleric acid (hereinafter abbreviated as 3HV) The method has been disclosed! (Patent Documents 6, 7). This P (3HB-co-3HV) is considered to be applicable to a wide range of applications because it is more flexible than P (3HB). As a matter of fact, P (3HB—co—3H V) actually increased the 3HV mole fraction! However, the change in physical properties is not so good, and the flexibility is not improved as much as required especially for films, etc., so it is used in the field of rigid molded products such as shampoo bottles and disposable razor handles. It was not used.

 [0013] In recent years, a two-component copolymer polyester (hereinafter, referred to as P (3HB-CO-3HH) t) of 3HB and 3-hydroxyhexanoic acid (hereinafter, abbreviated as 3HH) and a method for producing the same have been described. (See, for example, Patent Documents 8 and 9). The method for producing P (3HB-co-3HH) in these patent documents is a method for fermentative production of oils and fats such as fatty acids such as oleic acid by using aeromonas caviae isolated from soil. Met. Also, studies have been made on the properties of P (3HB-co-3HH) (see Non-Patent Document 6). In this report, A. caviae is cultured using fatty acids having 12 or more carbon atoms as the sole carbon source, and 3HH fermentatively produces 11 to 119 mol% of P (3HB-co-3HH). As P (3HB—CO-3HH) increases in mole fraction of 3HH, P (3HB) changes from hard and brittle properties to soft properties in turn, and P (3HB-CO-3HV) It was evident to show greater flexibility. However, with the above production method, the bacterial mass was 4 gZL, the polymer content was 30%, and the polymer productivity was low. Therefore, a method that could further increase the productivity for practical use was searched.

[0014] A polyhydroxyalkanoic acid synthase (hereinafter abbreviated as PHA synthase) gene has been cloned from Aeromonas' caviae, which produces P (3HB-co-3HH) ( See Patent Document 10 and Non-Patent Document 7). This gene was transformed into Ralstonia eutropha (formerly Alcaligenes eutrophus) and cultured using vegetable oil as a carbon source.As a result, the bacterial cell content was 4 g / L and the polymer content was 80%. Achieved (see Non-Patent Document 8). Also disclosed is a method for producing P (3HB-co-3HH) using a bacterium such as Escherichia coli or a plant as a host. The productivity is not described (for example, see Patent Document 11).

 [0015] The above polyester P (3HB-CO-3HH) has a wide range of properties from a hard polymer to a soft polymer by changing the mole fraction of 3HH, and thus requires hardness like a TV housing. It is expected to be applied to a wide range of fields, from those that require flexibility such as yarns and films. However, the productivity of P (3HB-co-3HH) is still low with the above-mentioned production method, and it is still insufficient as a production method for the practical use of P (3HB-co-3HH). I have to know.

 [0016] Several studies on the production of biodegradable polyester using yeast as a host have been reported. Leaf et al. Introduced a PHA synthase gene of R. eutropha into Saccharomyces cerevisiae, a type of yeast, to produce a transformant, and cultured it using glucose as a carbon source. Thus, accumulation of P (3HB) has been confirmed (see Non-Patent Document 9). However, the polymer content produced in the above study was only 0.5%, and the polymer was P (3H B), which was hard and brittle.

 [0017] It has also been studied to produce a PHA synthase gene derived from Pseudomonas aeruginosa in yeast Saccharomyces cerevisiae using fatty acids as a carbon source to produce a copolymer containing a monomer having 5 or more carbon atoms. . Also in this case, the content of the produced polymer was 0.5% (see Non-Patent Document 10).

 According to another study, along with the PHA synthase gene, β-ketothiolase, which synthesizes acetyl-CoA to synthesize 3-hydroxybutylyl-CoA, and NADPH-dependent reductase gene were introduced. It has been confirmed that 6.7% of the polymer is accumulated per body weight (Non-Patent Document 11). Under the pressure, these polymers were Ρ (3ΗΒ) with hard and brittle properties.

[0019] In addition, the yeast Pichia Pastoris (Pichia Pastoris) with peromokinome, pseudomona Studies have been conducted on the orientation expression of a PHA synthase gene derived from Pseudomonas aeruginosa and production of polyester using oleic acid as a carbon source. This study shows that it accumulates 1% by weight of polymer per dry cell (see Non-Patent Document 12). However, this level of polymer productivity is completely inadequate for industrial production.

 [0020] Yeasts are known to grow quickly and have high cell productivity. Among them, yeast belonging to the genus Candida has attracted attention as a single cell protein in the past, and production of feed cells using normal paraffin as a carbon source has been studied. In recent years, a host vector system of the genus Candida has been developed, and production of a substance using gene recombination technology has been reported (see Non-Patent Document 13). The productivity of α-amylase using Candida utilis as a host is as high as about 12.3 gZL. The genus Candida, which has such a high ability to produce substances, is expected as a host for polymer production. You. Furthermore, since the separation of bacterial cells and culture solution is easier than that of bacteria, it is possible to further simplify the process of polymer extraction and purification.

 Therefore, a method has been developed to produce P (3HB-CO-3HH), which has excellent physical properties, using yeasts of the genus Candida, but it is necessary to make further improvements in terms of polymer productivity. (See Patent Document 11). One of the ways to increase the amount of polymer produced per cell is to increase the intracellular expression of enzyme genes involved in PHA synthesis! A method was assumed.

[0021] Vectors, which are genes capable of autonomous replication in yeast cells, are classified into two types: a type in which about one copy exists in one cell (YCp type) and a type in which multiple copies can exist in one cell (YRp type). Is being developed. In Candida's maltosa, M. Kawamura et al. Reported that the transformation-causing region (Autonomously replicating sequence: ARS) and the centromeric sequence (CEN) by highly efficient transformation (Transformation ability). (Hereinafter abbreviated as TRA) (see Non-Patent Document 14), and a high-copy vector excluding the stable and low-copy vector having the entire TRA region and the CEN region where high expression of the transgene can be expected. Several vectors have been developed (see Non-Patent Document 15). However, high copy vectors such as Candida's maltosa have stability problems. And cannot be used industrially advantageously. Therefore, using high copy number vectors

It has been difficult to improve the productivity of polymers by increasing the expression level of the enzyme genes involved in PHA synthesis in cells.

[0022] As a method for increasing the amount of intracellular expression of an enzyme gene involved in PHA synthesis, a method of strengthening the promoter for expressing the gene can be considered. Various promoters have been cloned in Candida maltosa. Known as a glycolytic enzyme, a promoter of phosphodalicerate kinase (hereinafter abbreviated as PGK) is known to induce strong gene expression in the presence of glucose. Furthermore, a GAL promoter having a strong gene expression-inducing activity in the presence of galactose has also been cloned (see Non-Patent Document 16). However, these promoters hardly function when, for example, a fat or oil fatty acid or normal alkane (n-alkane) suitable for the production of P (3HB-CO-3HH) is used as a carbon source. Furthermore, since the GAL promoter is induced only when galactose is used as a carbon source, it is necessary to use expensive galatose, which means that it is suitable for industrial production.

[0023] Candida maltosa produces high levels of n-alkane oxidation enzymes in the presence of alkanes. In particular, a gene encoding cytochrome P450 (hereinafter abbreviated as ALK), which carries out the first acidification of alkanes, is strongly induced (see Non-Patent Document 17). However, these promoters also have lower activity compared to PGK and GAL promoters. Furthermore, promoters such as the actin synthase 1 gene (hereinafter, abbreviated as ACT1) that is constitutively expressed do not have sufficient strength for activity. As an example, if a fatty acid or fatty acid or n-alkane suitable for the production of P (3HB-CO-3HH) is used as the carbon source, at present, there are multiple ARR (alkane Responsible Region) sequences upstream of the ALK2 promoter. No promoter has been developed that is more powerful than the ARR promoter (see Non-Patent Document 18) whose promoter activity has been improved by the addition thereof. Therefore, a method for increasing the amount of intracellular expression of an enzyme gene involved in PHA synthesis has been developed. It is not practical to use a strong promoter.

[0024] Separately from this, a method of introducing a large number of expression units of an enzyme gene involved in PHA synthesis into a vector can be considered. Is known to be limited by the size of the transgene, and vectors containing genes of too large a size are difficult to construct, transfer efficiency into yeast, and stability in yeast. It is difficult to say that it is industrially realistic.

 [0025] In addition, a method for increasing the activity of a target enzyme by amplifying a gene has been reported (see Non-Patent Document 19). In this method, a cycloheximide-resistant gene and a target gene are linked and introduced into a cycloheximide-sensitive yeast to obtain a high-concentration cycloheximide-resistant strain, thereby obtaining a strain with high expression of the target gene. . However, Candida maltosa is known to be resistant to cycloheximide, and such gene amplification using cycloheximide-resistant genes allows the transfer of enzymes involved in PHA synthesis. It is difficult to increase the amount of expression in the bacterial cells of the offspring.

 [0026] Therefore, it has been desired to develop a method for avoiding the above-described problems and increasing the intracellular expression level of an enzyme gene involved in PHA synthesis in Candida 'maltosa to produce high biodegradable polyester. .

 [0027] Further, it is generally known that the molecular weight of the polyester greatly affects the physical properties and the curability. In the production of PHA in microorganisms, it is known that if the number of enzyme molecules per cell is excessively increased, a substrate-limited state is reached, and the molecular weight of the produced polymer decreases (see Non-Patent Documents 20 and 21). Therefore, it has been desired to develop a method for controlling the molecular weight of the polyester produced in the cells. It is also known that when the produced polyester is a copolymer, the monomer composition greatly affects the physical properties and processability. Therefore, development of a method for controlling the monomer composition of the copolymerized polyester has been desired.

[0028] In addition, in order to breed a PHA-high producing strain, it is necessary to increase the intracellular expression level of an enzyme gene involved in PHA synthesis, and to produce a PHA produced by a transformed strain into which the gene group has been introduced. In some cases, it is necessary to introduce the same gene group in consideration of the properties and physical properties. Usually, in obtaining a transformant, markers such as drug resistance and auxotrophy are used. For this reason, it is necessary to use different types of markers according to the number of gene introductions.Candida's maltosa, which has multiple gene markers developed to date, is significantly inferior to the wild type in its growth rate, As a PHA producer It was difficult to use (see Non-Patent Document 2). In addition, Candida maltosa having one type of genetic marker and improved in growth rate has also been developed (see Patent Document 5). It is considered difficult to add multiple gene markers while maintaining the growth rate of this strain while maintaining the same growth rate as that of the wild type strain, because the yeast has a diploid genome. Was done.

Patent Document 1: JP-A-62-74287

Patent Document 2: JP-A-62-74288

Patent Document 3: International Publication No. 99Z04014 pamphlet

Patent Document 4: International Publication No.01Z88144 pamphlet

Patent Document 5: JP-A-2002-209574

Patent Document 6: JP-A-57-150393

Patent Document 7: JP-A-59-220192

Patent Document 8: JP-A-5-93049

Patent Document 9: JP-A-7-265065

Patent Document 10: JP-A-10-108682

Patent Document 11: WO00Z43523 pamphlet

Non-Patent Document 1: Non-Conventional Yeasts in Biotechnology.A edited by Wolf K.

Handbook, Springer ~ Verlag, Berlin (1996) p411-580)

Non-Patent Document 2: Kawai S. et al., Agric. Biol. Chem., 55: 59-65 (1991) Non-Patent Document 3: Takagi et al., J. Gen. Appl. Microbiol., 31: 267-275 (1985) Non-Patent Document 4: Ohama T. et al., Nucleic Acid Res., 21: 40394045 (1993) Non-Patent Document 5: M. Lemoigne, Ann.Inst. Pasteur, 39, 144 (1925)

Non-Patent Document 6: Y. Doi, S. Kitamura, H. Abe, Macromolecules, 28, 4822-4823 (1995)

Non-Patent Document 7: T. Fukui, Y. Doi, J. Bacteriol, vol. 179, No. 15, 4821-4830 (1997)

Non-patent document 8: T. Fukui et al., Appl. Microbiol. Biotecnol. 49, 333 (1998) Non-patent document 9: Microbiology, vol. 142, ppl 169—1180 (1996) Non-Patent Document 10: Poirier Y. et al., Appl. Microbiol. Biotecnol. 67, 5254-5260 (2001)

Non-patent literature ll: Breuer U. et al., Macromol. Biossci., 2, pp380-386 (2002) Non-patent literature 12: Poirier Y. et al., FEMS Microbiology Lett., Vol. 207, pp97-102 (2002)

 Patent Document 13: Danigaku and Biology, vol. 38, No9, 614 (2000)

Non-Patent Document 14: M. Kawamura, et al., Gene, vol. 24, 157, (1983) Non-Patent Document 15: M. Ohkuma, et al., Mol.Gen. Genet., Vol. 249, 447, (19

95)

Non-Patent Document 16: S. M. Park, et al., Yeast, vol. 13, 21 (1997)

Non-Patent Document 17: M. Ohokuma, et al., DNA and Cell Biology, vol. 14, 16

3, (1995)

Non-Patent Document 18: Kogure, 2002 Abstracts of Japanese Society of Agricultural Chemistry, pl91

Non-Patent Document 19: K. Kondo, et al., Nat.Biotechnol., Vol. 15, pp 453-457 (

1997)

Non-Patent Document 20: Sim S.J. et al., Nature Biotechnology, vol. 15, pp63-67 (1997).

Non-Patent Document 21: Gerngross T.U., Martin D.P., Proc.Natl.Acad.Sci.U SA, vol. 92, pp6279-6283 (1995)

Disclosure of the invention

Problems to be solved by the invention

The present invention has been made in view of the above-mentioned current situation, and it is possible to introduce a large number of various genes by constructing a multiple auxotrophic gene-disrupted strain in yeast, particularly in the genus Candida, and to efficiently produce a useful substance. In view of the above situation, the present invention also provides a transformant obtained by transforming a plurality of expression cassettes of genes involved in PHA synthesis into an enzyme, and By culturing the transformed transformant, a polyester such as P (3HB-CO-3HH) having biodegradability and excellent physical properties can be produced. To provide a way to: The present invention also provides a method for breeding the microorganism. Means for solving the problem

 The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, by making full use of genetic recombination techniques, the yeast phosphoribosylaminoimidazole-succinocarboxamide synthase (EC6 3.2.6) DNA encoding (ADE1 gene), DNA encoding histidinol-phosphate aminotransferase (EC 2.6.1.9) (HIS 5 gene), and orotidine 5, monophosphate decarboxylate Production of ADE1, HIS5 and URA3 gene disrupted strains by homologous recombination with chromosomal DNA using the DNA (URA3 gene) fragment encoding the race (EC 4.1.1.23) And succeeded in obtaining a gene-disrupted yeast requiring adenine, histidine and peracil. Then, the growth ability of the gene-disrupted yeast was compared to AC16 of the ADE1 gene-disrupted strain of Candida maltosa, and it was found that the growth ability was superior to that of CHA1 of the ADE1 gene-mutated strain by mutation treatment of Candida maltosa. The present inventors have completed the present invention by showing that they are equivalent to the clarified AC16 strain.

 The present inventors have also conducted intensive studies to solve the above-mentioned problems. As a result, the gene recombination technique was used to obtain polyhydroxyalkanoate synthase gene (hereinafter abbreviated as phaC) and acetoacetyl CoA. Transformants were prepared by introducing a plurality of reductase genes (EC 1. 1. 1. 36) (hereinafter abbreviated as phbB) into gene-disrupted strains of Candida maltosa. As a result, a two-component copolymerized polyester (hereinafter abbreviated as 3HH) and 3-hydroxybutyrate (hereinafter abbreviated as 3HB) and 3-hydroxyhexanoate (hereinafter abbreviated as 3HH) useful as a biodegradable polyester can be obtained. , P (abbreviated as 3HB-co—3HH) was successfully produced.

That is, the first present invention relates to a yeast in which the URA3 gene of chromosomal DNA has been disrupted by homologous recombination with a URA3 DNA fragment; the HIS5 gene of chromosomal DNA has been homologously recombined with a HIS5 DNA fragment. For destroyed yeast. Similarly, the present invention relates to a yeast in which both the ADE1 gene and the URA3 gene have been disrupted; a yeast in which both the ADE1 gene and the HIS5 gene have been disrupted; a yeast in which both the URA3 gene and the HIS5 gene have been disrupted; and the ADE1 gene and the URA3 gene. It relates to yeast in which the HIS5 gene has been disrupted. Further, the present invention relates to a transformant of the above-described gene-disrupted yeast transformed with a DNA sequence containing a homologous or heterologous gene.

Further, the present invention provides a method for producing an industrially useful substance by obtaining a transformant in which a plurality of heterologous gene expression systems have been introduced into the gene-disrupted strain. Specifically, as an example, 3-hydroxybutyrate (hereinafter abbreviated as 3HB) and 3-hydroxyhexanoate (hereinafter abbreviated as 3HB), which are useful as a biodegradable polyester, are added to the gene disrupted Candida maltosa strain of the present invention. , 3HH) and a polyhydroxyalkanoic acid synthase gene (hereinafter abbreviated as phaC) which is an enzyme that synthesizes a two-component copolymerized polyester (hereinafter abbreviated as P (3HB-CO-3HH)). P (3HB-co-3HH) can be efficiently produced using the transformant into which acetoacetyl CoA reductase gene (EC1.1.1.36) (hereinafter abbreviated as phbB) is preferably introduced. Was successfully produced.

 That is, the present invention is not only a method for producing a gene expression product (especially, polyester) using a gene-disrupted yeast, but also a method for producing a polyester using a transformant into which a plurality of genes involved in polyester biosynthesis have been introduced. Further, the present invention is also a method for producing a polyester, wherein the polyester is collected from a culture obtained by culturing the above transformant.

[0034] Furthermore, the present invention is a method for producing a polyester in which the physical properties of the produced polyester are controlled. The present invention also relates to a method for efficiently recovering a selection marker used for gene transfer.

 [0035] That is, the second present invention is a yeast transformant into which a polyhydroxyalkanoic acid synthase gene and an acetoacetyl CoA reductase gene have been introduced. This is a yeast transformant characterized in that two or more copies have been introduced.

 The present invention also relates to a method for producing a polyester using the yeast transformant, wherein the polyester is collected from a culture obtained by culturing the yeast transformant. is there.

The present invention is also a method for controlling the molecular weight of the polyester by controlling the number of acetoacetyl CoA reductase genes in the yeast transformant in the production of polyester using the yeast transformant. The present invention is also a method for controlling the number of polyhydroxyalkanoic acid synthase genes in a yeast transformant in the production of a polyester using the yeast transformant, thereby controlling the hydroxyalkanoic acid composition of the polyester. .

 The present invention also provides a method for recovering a selectable marker, which comprises removing the selectable marker gene by performing intramolecular homologous recombination with Candida maltosa having the selectable marker gene.

 Hereinafter, the present invention will be described in detail.

 First, examples of the gene-disrupted yeast of the present invention include the following.

 Peracil-requiring gene-disrupted yeast in which the URA3 gene of chromosomal DNA has been disrupted by homologous recombination with a URA3 DNA fragment;

 A histidine-requiring gene-disrupted yeast in which the HIS5 gene of chromosomal DNA has been disrupted by homologous recombination with a HIS5 DNA fragment;

 Adenine and peracil-requiring gene-disrupted yeast in which the A DEI gene and URA3 gene of chromosome DNA have been disrupted by homologous recombination with the ADE1 DNA fragment and URA3 DNA fragment;

 Adenine- and histidine-requiring gene-disrupted yeast in which the ADEI gene and the HIS5 gene of chromosome DNA have been disrupted by homologous recombination with the ADE1 DNA fragment and the HIS5 DNA fragment;

 A URA3 and histidine-requiring gene-disrupted yeast in which the URA3 gene and the HIS5 gene of chromosomal DNA have been disrupted by homologous recombination with the URA3 DNA fragment and the HIS5 DNA fragment;

 A gene-disrupted yeast requiring adenine, peracil, and histidine in which the ADE1 gene, URA3 gene, and HIS5 gene of chromosomal DNA have been disrupted by homologous recombination with the ADE1 DNA fragment, URA3 DNA fragment, and HIS5 DNA fragment.

[0037] For yeast that disrupts the ADE1, URA3, and HIS5 genes by homologous recombination, yeast that has been deposited with a strain depository (eg, IFO, ATCC, etc.) is used without any particular limitation. Can be. Preferably, in terms of assimilation of hydrophobic substances such as linear hydrocarbons, the genus Candida, the genus Clavispora, and the varieties of Calispora are used. Genus Cryptococcus, Genus Denoyomyces (Genus Debaryomyces), Genus Roderomyces (Genus Lodderomyces), Genus Metschnikowia (Genus Metschnikowia), Genus Pichia (Genus Pic hia), Genus Rhodosporidium (Genus Rhodosporidium) ), Yeasts of the genus Rhodotorula, genus Sporidiobolus, genus Stephanoascus (genus Stephanoascus) and genus Yarrowia can be used.

 Among these yeasts, the genus Candida is more preferable, in particular, since the analysis of the chromosomal gene sequence has been advanced, a host-vector system can be used, and the ability to assimilate linear hydrocarbons and oils and fats is high.

 [0038] Among the genus Candida, albi cans¾, ancuaensis¾ ^ atmospnaenca, azyma, bertae, blankii¾, butyri, conglobata, dendronema Species, ergastensis, fluviatilis, friearich 11, gropengiesseri, haemulonu, incommunis, insectrum, laureli, maltosa ^ a ^ melibiosica, membmnifaciens, me s enteric a¾ ^ natal ensis, oregonolea, palmiole Species, parapsilosis, psudo intermedia ia, quercitrusa, rhagu ^ a, rugosa, saitoana, saketan, schatavii, seq uanensis ^ a ^ shehatae®, sorbophila, tropicalis, valdiviana, viswanat hii, etc. More preferred to use.

 Among these species, maltosa is particularly preferred because of its high growth rate when a straight-chain hydrocarbon is used as a carbon source and high safety, unlike albicans.

[0039] Further, with respect to the production and use of the ADE1, URA3, and HIS5 gene-disrupted strains of the present invention, Candida's maltosa can be used as a preferred example of yeast.

 The Candida 'Maltosa AC16 strain, which is the ADE1 gene disrupting yeast used in the present invention, has the accession number of FERM BP-7366, and, as of November 15, 2000, 1-1 1-1 Tsukuba East, Ibaraki, Japan 1 It has been internationally deposited at the Patent Organism Depositary of the National Institute of Advanced Industrial Science and Technology (AIST) in the Central No. 6 based on the Budapest Treaty.

 In addition, each gene disrupted yeast obtained in the present invention as described below, that is,

Candida maltosa U-35 strain, a URA3 gene disrupting yeast (Accession No. FERM P-19435, deposited on July 18, 2003), HIS 5 gene disrupting yeast Candida maltosa CH-I strain (Accession No. FERM P 19434, deposited on July 18, 2003),

 ADE1 and URA3 gene disrupting yeast strain Candida maltosa UA-354 strain (Accession No. FERM P-19436, deposited on July 18, 2003),

 ADE1 and HIS5 gene-disrupted yeast, Candida 'maltosa AH-15 strain (Accession No. FERM P-19433, deposited on July 18, 2003),

 Candida maltosa HU-591 strain, which is a HIS5 and URA3 gene disrupting yeast (Accession number: FERM P-19545, deposited: October 1, 2003),

 ADE1 and HIS5 and URA3 gene disrupting yeast, Candida maltosa AHU-71 strain (Accession No. FERM BP-10205, deposited on August 15, 2003),

 Are deposited with the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary, located at 1-1, Tsukuba-Higashi, Ibaraki, Japan.

[0040] Here, homologous recombination refers to recombination that occurs in a portion where the base sequence of DNA is similar or has the same sequence (homologous sequence).

 Gene disruption refers to mutation of the base sequence of a gene, insertion of another DNA, or deletion of a portion of the gene so that the function of the gene cannot be performed. As a result of gene disruption, the gene cannot be transcribed into mRNA, and the structural gene is not translated, or the transcribed mRNA is incomplete, resulting in mutation or deletion in the amino acid sequence of the translated structural protein. The function of is impossible.

 [0041] The ADE1 gene refers to a 5 'untranslated region including a promoter region, a region encoding phosphoribosylaminoimidazo-l-succinocarboxamide synthase (EC 6.3.3.2.6), and a terminator region. The gene fragment containing the 3 'untranslated region is shown. The nucleotide sequence of the Candida maltosa ADE1 gene has been published in GenBank: D00855.

[0042] The URA3 gene refers to a 5, untranslated region including a promoter region, a region encoding orotidine 5, -phosphate decarboxylace (EC 4.1.1.23), and a non-translated region including one terminator. 1 shows a gene fragment consisting of a region. Candida Maltosa The nucleotide sequence of the URA3 gene has been published in GenBank: D12720.

 [0043] The HIS5 gene includes a 5 'untranslated region including a promoter region, a region encoding histidinol-phosphate aminotransferase (EC 2.6.1.9), and a 3' region including a terminator region. 1 shows a gene fragment consisting of an untranslated region. The nucleotide sequence of the Candida maltosa HIS5 gene has been published in GenBank: X17310.

 [0044] The ADE1 DNA fragment refers to a DNA capable of causing homologous recombination with the ADE1 gene on the chromosome in a microbial cell, thereby destroying the ADE1 gene. A URA3 DNA fragment refers to a DNA that can cause homologous recombination with the URA3 gene on the chromosome in a microbial cell, thereby destroying the URA3 gene! A HIS5 DNA fragment refers to a DNA that can undergo homologous recombination with the HIS5 gene on the chromosome in a microbial cell, thereby destroying the HIS5 gene!

 Next, as the transformant of the present invention, the above-described gene-disrupted yeast is transformed with a DNA sequence containing a homologous or heterologous gene.

 [0046] The homologous gene means a gene present on the chromosome of the host yeast or a partial DNA thereof. A heterologous gene refers to a gene that is not originally present on the chromosome of the host yeast or a partial DNA thereof.

 [0047] A gene expression cassette can also be used. A gene expression cassette is a circular plasmid composed of a DNA sequence containing a transcription promoter DNA sequence, DNA encoding a gene to be expressed, and a terminator that terminates transcription. And those that integrate into chromosomal DNA.

 Next, as a method for producing a gene expression product of the present invention, a homologous or heterologous gene expression product is collected from a culture obtained by culturing the above transformant. The gene expression product is particularly preferably polyester.

 [0049] The gene expression product is itself a gene expression product when the substance expressed by the gene (gene expression product) is a desired protein or enzyme. In addition, the gene expression products are various enzymes and coenzymes, and substances that are produced by the enzymes expressing catalytic activity in the host yeast and are directly different from the gene expression products are also gene expression products. It is.

[0050] PHA is an abbreviation for polyhydroxyalkanoate, and is a copolymer of 3-hydroxyalkanoic acid. 2 shows a combined, biodegradable polyester.

 phaC indicates a polyhydroxyalkanoic acid synthase gene that synthesizes a biodegradable polyester copolymerized with 3-hydroxyalkanoic acid.

 phbB refers to an acetoacetyl CoA reductase gene that reduces acetoacetyl CoA to synthesize 3-hydroxybutylyl-CoA.

[0051] Hereinafter, a method for producing an ADE1, URA3, and HIS5 gene-disrupted strain and a method for producing a polyester using the disrupted strain will be specifically described.

 (1) Method for producing URA3 gene-disrupted strain, URA3 / ADE1 gene-disrupted strain

 Regarding the production of a URA3 gene disrupted strain, such a method can be used if a disrupted strain that does not express the URA3 enzyme is obtained. Although various methods for gene disruption have been reported, gene disruption by homologous recombination is preferable because only a specific gene can be disrupted (Nickoloff JA, edited in Methods in Molecular Biology, 47: 291-). 302 (1995), Humana Press Inc., Totowa ゝ NJ)). Among homologous recombinants, a disrupted strain that does not return spontaneously can be obtained, and as a result, when a strain is obtained with high safety in handling a recombinant, gene replacement disruption is preferred in terms of obtaining a strain.

[0052] As the URA3 DNA fragment to be used, a DNA fragment in which a partial DNA inside a gene is removed and the remaining both ends are ligated again is used.

 The part of DNA to be removed is a part where the URA3 gene can no longer exhibit enzymatic activity due to the removal, and has a length that does not restore the URA3 enzyme activity due to spontaneous reversion. Although the chain length of such partial DNA is not particularly limited, it is preferably 50 bases or more, more preferably 100 bases or more. Also, DNA of any length may be inserted into the removed DNA site!

[0053] These DNA fragments can be prepared by, for example, PCR (polymerase chain reaction), excision from a vector with a restriction enzyme, and religation.

 The length of the homology region at both ends of the URA3 DNA fragment is preferably at least 10 bases, more preferably at least 200 bases, and even more preferably at least 300 bases.

Further, the homology of each end is preferably 90% or more, more preferably 95% or more. [0054] It was expected that two or more URA3 genes would be present in the Candida maltosa chromosome. A selection marker is not necessary when there is a method that can detect that the target disrupted gene has been disrupted.However, when two or more genes are disrupted in a chromosome, the selection marker is usually used as an indicator for the yeast chromosome. It is necessary to detect the homologous recombination of the gene to be destroyed. The force of using multiple selectable markers, or the work of destroying the first gene and then removing or destroying the used selectable marker gene, and then destroying the second and subsequent genes is required.

 [0055] Therefore, a method of inserting a gene that can be a selection marker such as ADE1 into the gene portion from which the URA3 DNA fragment has been removed can be used. The length of the selection marker to be inserted is not particularly limited as long as it includes a promoter region, a structural gene region, and a terminator region that can function substantially in yeast. It does not work even if the genetic marker is derived from an organism different from the target yeast.

 Further, a hisG gene fragment (a fragment of the ATP phosphoribosyl transferase gene of Salmonella, a plasmid ρ 遺 伝 子 09 containing this gene fragment is available from the ATCC (ATCC: 87624)) is inserted into both ends of the selectable marker gene, respectively. After gene disruption, the inserted marker gene can be removed by intramolecular homologous recombination (Alani et al., Genetics, 116: 541-545 (1987)). As described above, there is no particular restriction on the gene fragment used for removing the selectable marker gene, and homologous fragments of any gene may be arranged above and below the selectable marker. Therefore, it is also possible to use the sequence contained in the selectable marker. In the present invention, the molecular fragment of the ADE1 gene 5, which is used as a marker, is linked to the end of the ADE1 gene 3, so that the molecule can be used. The internal homologous recombination has made it possible to remove the marker gene very efficiently. There is no particular restriction on the gene fragment used for intramolecular homologous recombination of the marker. A gene fragment which does not substantially function as a marker gene should be used. 3. Gene fragments at the end can also be used.

 [0057] In the present invention, three types of URA3 disrupting DNA were used.

The DNA-1 for URA3 disruption is a DNA obtained by removing a DNA fragment encoding the URA3 enzyme of about 220 bp and ligating a DNA fragment of about 350 bp on the 5 ′ side and a DNA fragment of about 460 bp on the 3 ′ side (see FIG. The portion removed from Do accounts for about 30% of the URA3 enzyme protein. The homology between the 5′-side and 3′-side DNA fragments and the original URA3 gene is 100% for both DNAs.

 [0058] In the DNA-2 for URA3 disruption, the ADE1 gene derived from Candida maltosa was inserted instead of the URA3 DNA fragment from which the DNA-1 for URA3 disruption had been removed (Fig. 1).

 The DNA-3 for URA3 disruption uses about 630 bp of the 5'-terminal part of the ADE1 gene as a sequence for causing intramolecular homologous recombination and restoring adenine requirement, and is connected downstream of the ADE1 gene. (Fig. 1).

 [0059] The DNA fragment used in the present invention can be constructed on a general vector.

 In the present invention, pUC-Nx was used. pUC—Nx is the DNA between the EcoRI and HindI sites of pUC19 (Edited by Sambrook et al., Molecular cloning: A Laboratory Manual ^ Second Edition I. ld, Cold Spring Harbor Laboratory Press (1989)), as SEQ ID NO: 1. This is a vector in which a restriction enzyme site has been newly constructed by substitution with the described DNA.

 From pUC 119-URA3 (Ohkuma M. et al., Curr. Genet., 23: 205-210 (1993)), PCR was used to separately amplify the 5 'and 3' sides of the URA3 gene to form pUC-Nx Ligation was performed sequentially to prepare a vector containing DNA-1 for URA3 disruption.

 Next, the ADE1 gene amplified by the PCR method was inserted into the vector, and a vector containing DNA-2 for URA3 disruption was prepared in this way.

 Further, by inserting about 630 bp of the ADE15 'terminal partial sequence amplified by PCR into the ADE1 gene 3' end of this vector, a vector containing URA3 disrupting DNA-3 capable of removing the marker gene was prepared.

[0060] The vector containing the DNA for disruption is introduced into an appropriate Escherichia coli, for example, «JM109 or DH5a, and the Escherichia coli is cultured. sambrook et al., Molecular cloning: A Laboratory Manual ^ Second Edition 1.42-1.47, Cold Spring Harbor Laboratory Press (198 9)) ₀ Alternatively, it is possible to use an alkali method or the like (Brinbioim HC, etc.). Nucleic Acids Res. 7: 1513-1523 (1979)). It is also possible to use a commercially available plasmid purification kit or the like. This vector can be used directly for gene disruption, but It is desirable to cut out a homologous portion containing the URA3 region from the vector with an appropriate restriction enzyme, and use that as the DNA for disruption. It is also possible to amplify using the PCR method. In the present invention, the URA3 gene could be disrupted by homologous recombination by cutting with restriction enzymes Sphl and Swal and introducing the DNA fragment into the cells without purification.

 [0061] Candida * maltosa transformation methods include the protoplast method, lithium acetate method (Taka gi M. et al., J Bacteriol, 167: 551-5 (1986)), and electric pulse method (Kasuske A. et al. : 691-697 (1992)) The known force is used in the present invention. Commercial equipment can be used to generate electric noise. In the present invention, ELECTRO CELL MANIPULATOR 600 manufactured by BTX (San Diego, CA USA) was used. The cuvette used was BM6200 (2 mm gap blue cap) manufactured by BIO MEDICAL CORPORATION CO. LTD (Tokyo Japan).

 Competent cells were prepared from 16 AC strains, and after electropulsing with DNA-2 for URA3 disruption, cultured in an adenine-free medium, and from the resulting colonies, the disrupted strain in which the target URA3 gene was inserted into the target URA3 gene was obtained. Screen.

 [0062] Screening of the target gene-disrupted strain is performed by PCR from the obtained colonies using the Genomics Southern Hybridization method (edited by Sambrook et al., Molecular cloning: A Laboratory Manual ^ Second Edition 9.3-9-1. 57, old spring Harbor Laboratory Press (1989)). In the PCR method, when both ends of the URA3 gene are used as primers, in agarose gel electrophoresis, a normal size DNA band is detected in a wild type strain, and a large band is detected in a force-disrupted strain by the amount of the inserted gene. In the present invention, DNA bands of about lkbp and 2 kbp were detected. However, in the case of substitution or integration with chromosomal DNA such as gene disruption, it is necessary to assume that the gene may be inserted into a part other than the target, for example, an unknown part with high homology. May not be confirmed. In this case, it can be confirmed by performing a genomic Southern hybridization method or a PCR method using a gene sequence existing outside the portion used for homologous recombination of the gene to be disrupted.

[0063] It is expected that two or more URA3 genes are present on the Candida * maltosa chromosome. Was. In fact, the strain obtained by transforming the DNA-2 for URA3 disruption does not exhibit the requirement for peracil, and unless the second URA3 gene is disrupted, it will not be able to confer the requirement for peracil. In order to disrupt the second URA3 gene, disrupt the inserted ADE1 gene and then use the technique of disrupting the first URA3 gene again, or after transforming with URA3 disrupting DNA-1 etc. This can be achieved by selecting colonies that grow in the presence of peridine, peridine or peracil and 5-FOA (5-Fluoro-Orotic-Acid). In the present invention, the former method is used. That is, URA3 disrupting DNA-2 was transformed into URA3 disrupting strain, and URA3 disrupting DNA-1 was electrotransferred into the strain that became non-adenine-requiring, and the first URA3 gene was disrupted to contain adenine. Adenine-requiring strains can be obtained by applying to a minimal medium and selecting the red colonies that appear. An ADE1 DNA fragment can also be used (JP-A-2002-209574). Then, work to destroy the second URA3 gene.

 At this time, it is preferable to use a concentration step for performing screening efficiently. For example, a method called nystatin enrichment (Snow R. Nature 211: 206-207 (1966)) can be used. This method has been developed to efficiently select mutant strains obtained by random mutation in yeast, but can also be applied to gene-disrupted strains. For example, after gene transfer, the cultured cells are inoculated into YM medium or the like and cultured. After washing the bacteria and culturing in a minimal medium without a nitrogen source, cultivate briefly in a minimal medium with a nitrogen source. Wild strains can be preferentially killed by adding nystatin directly to this culture and aerobically culturing at 30 ° C for 1 hour. This bacterial solution is spread on an appropriate agar plate containing adenine and cultured at 30 ° C for about 2 days to obtain red colonies.

 [0065] The obtained adenine-requiring strain can be confirmed by a PCR method. When both ends of the URA3 gene are used as primers, agarose gel electrophoresis can detect DNA bands of normal size in the original strain. In the ADE1-disrupted strain, a band shorter than the deletion is also detected.

[0066] Next, one strain of this URA3 gene was disrupted, and the above-mentioned method was repeated for a strain in which adenine auxotrophy was restored, whereby the second URA3 gene was disrupted to become peracilotropic. You can get shares. After that, double nutrition is required by destroying the ADE1 gene again. Ability to Obtain Requirement Strain In the present invention, the disruption of the second URA3 gene includes the URA3 disruption DNA-3 containing the ADE1 gene, which can remove the marker gene by intramolecular homologous recombination. Was used. The disrupting gene is electrotransformed into the strain and colonies are formed in a selective medium containing peridine or peracil. The resulting colonies are replicated on a medium containing no lysine peracil to select periracil-requiring strains. The obtained chromosome gene of the auxotroph is analyzed by PCR, etc., and the gene corresponding to the normal URA3 is not amplified, and only the gene containing the inserted gene and the gene containing the deletion are amplified. Select a stock. At this stage, the URA3-disrupted strain is completed.

 Next, the inserted ADE1 gene is removed. As this method, a strain in which the ADE1 gene has been naturally deleted by intramolecular homologous recombination can be easily prepared by applying the nystatin enrichment method. According to a preferred embodiment of the present invention, the cultured cells are inoculated on a YM medium or the like, cultured, washed, cultured in a minimal medium without a nitrogen source, and then cultured in a minimal medium with a nitrogen source for a short time. By directly adding nystatin to this culture and aerobically culturing at 30 ° C for 1 hour, the strain containing the ADE1 gene can be preferentially killed. When this bacterial solution is spread on a suitable agar plate containing adenine and cultured at 30 ° C for about 2 days, red colonies are obtained.

 [0068] The obtained adenine-requiring strain can be confirmed by a PCR method or the like. When both ends of the URA3 gene were used as primers, agarose gel electrophoresis detected a DNA band with the inserted size of the ADE1 gene and the ADE1 fragment gene and a DNA with the size of the URA3 gene with a deletion in the original strain. In the ADE1-disrupted strain, a DNA band of the size of the URA3 gene with the deletion and a DNA band of the size of the ADE1 gene fragment added to the DNA of the size of the URA3 gene with the deletion are also detected. Is done. At this stage, ADE1 and URA3 gene disrupted strains are produced.

 (2) Method for producing HIS5 gene-disrupted strain, HIS5-ADE1 gene-disrupted strain

 HIS5 gene-disrupted yeast and HIS5'ADE1 gene-disrupted yeast can also be prepared from the AC16 strain using the method described in (1) above.

The HIS5 gene to be used is pUC119-HIS5 (Hikiji. Et al., Curr. Genet., 16: 261-2. 66 (1989)). That is, HIS5 gene disruption DNA or the like in which the 5 'side DNA fragment of the HIS5 gene and the 3' side DNA fragment are linked to both ends of a gene in which the 5 'side fragment of the ADE1 gene is connected to the 3' side of the ADE1 gene. (Figure 1). In the present invention, the ability to use a DNA fragment of about 500 bp on the 5 ′ side and 3 ′ side of the HIS5 gene is not particularly limited.

 [0070] The above gene was electrotransformed into 16 strains of AC, and a strain in which the HIS5 gene was disrupted was selected from the obtained adenine non-auxotrophs by PCR or the like, and then homologous intramolecularly by nystatin enrichment. A strain whose adenine requirement has been restored by recombination can be easily obtained. When there are a plurality of HIS5 genes, adenine-histidine double auxotrophs can be obtained by repeating this step.

 (3) Method for producing URA3 'HIS5 gene-disrupted strain, URA3' HIS5 'ADE1 gene-disrupted strain

 Based on the URA3'ADE1 disrupted strain obtained in (1), a URA3-HIS5 gene disrupted strain and a URA3'HIS5'ADE1 gene disrupted strain can be prepared by the method described in (2). . Alternatively, it can be prepared using the method of (1) based on the strain obtained in (2).

 (4) Expression of heterologous gene by gene-disrupted strain

 Using the gene-disrupted strain obtained in the present invention, a homologous gene or a heterologous gene can be introduced multiple times depending on the number of available markers, or can be introduced repeatedly by recovering the markers. Thus, the target gene can be introduced more than ever before and can be expressed in large amounts.

 Since yeast can secrete a glycosylated protein, which cannot be produced by Escherichia coli, into a medium, such a protein can be produced using a gene-disrupted strain. Further, since the yeast of the present invention has a plurality of gene markers, it can express several kinds of proteins, and can perform complicated reactions involving a plurality of enzymes. It is also useful for manufacturing.

The homologous gene that can be introduced is not particularly limited.For example, by introducing a P450 enzyme gene derived from the same strain into Candida's maltosa as disclosed in WO99Z04014 as a production example of an industrially useful product. , The production of dicarboxylic acids Further, the heterologous gene is not particularly limited, and examples thereof include production of the protein by introducing an antibody gene, a lipase gene, an amylase gene, and the like. Polyester production by introducing a polyhydroxyalkanoic acid synthase gene or an enzyme gene for synthesizing a substrate for polyhydroxyalkanoic acid synthesis is also included.

 [0073] The number of target genes to be introduced per yeast cell is determined by the properties of the target gene product and the strength of the promoter used. For example, in the case of a protein translated from an expression cassette into which a target gene product has been introduced, any number of simple proteins may be used, but in the case of a protein to which a sugar chain is added, translation of an excess protein is rate-limited by sugar chain modification. Which gives a heterogeneous product. Therefore, the introduction of a limited number of expression cassettes is preferred.

 [0074] In some Candida yeasts such as Candida maltosa used in the present invention, at the stage where protein is translated from mRNA, the manner in which some codons are translated differs from that of other organisms. It is known. In Candida's maltosa, the leucine codon CUG is translated into serine (Ohama T. et al., Nucleic Acid Res., 21: 40394045 (1993)), so the lacZ gene from E. coli is translated into active j8 galactosidase. No (Sugiyama H. et al., Yeast 11: 43-52 (1995)). When expressing a heterologous gene in this way, there is no guarantee that it will be translated into a functional protein in Candida maltosa. Therefore, when expressing a heterologous gene using Candida maltosa as a host, only leucine codon should be converted in principle, but other amino acid codons may be adjusted to those of Candida maltosa for more efficient expression. !,. The codon conversion may be performed with reference to, for example, Candida maltosa p524-527, by Mauersberger S. in Nonconventional Yeasts in Biotechnology, edited by Wolf K.

 [0075] In a preferred embodiment of the present invention, a biodegradable polyester is produced as a gene expression product. Hereinafter, a method for producing polyester will be described.

In the present invention, for example, an enzyme gene involved in polyester synthesis such as a polyhydroxyalkanoic acid synthase gene (phaC) or an enzyme gene involved in the synthesis of a molecule serving as a substrate for polyester synthesis is replaced with the above-described gene-disrupted yeast. Into a transformant, and collect the polyester from the culture obtained by culturing the transformant. [0076] The enzyme gene involved in polyester synthesis is not particularly limited, but is preferably an enzyme gene involved in synthesis of a polyester obtained by copolymerizing 3-hydroxyalkanoic acid represented by the following general formula (1). Involved in the synthesis of copolymerized polyester P (3HB—co—3HH) obtained by copolymerizing 3-hydroxybutyric acid represented by the following formula (2) and 3-hydroxyhexanoic acid represented by the following formula (3) More preferably, it is an enzyme gene.

 [0077] [Formula 1]

 [0078] [Formula 2]

CH ₃

 HO— CH ~ CH Riichi C——OH (2)

 II

 o

[0079] [Formula 3]

[0080] For example, the polyester synthase gene described in JP-A-10-108682 can be used.

 [0081] In addition to the present polyester synthase gene, a gene relating to (R) -3-hydroxybutylyl-CoA or (R) -3-hydroxybutylylhexanoyl-CoA, which is a substrate for polyester synthesis, was introduced. Is also good.

These genes include, for example, (R) -form-specific enolyl CoA hydratase (Fukui), which converts enolyl CoA, an intermediate of the j8 oxidation pathway, to (R) -3-hydroxyacyl-CoA. T. et al., FEMS Microbiology Letters, 170: 69-75 (1999), JP-A-10-108682), 13 ketothiolase that dimerizes acetyl-CoA to synthesize 3-hydroxybutylyl-CoA, depends on NADPH Sex reductase gene (Peoples OP et al., J. Biol. Chem. 264: 15298-15303 (1989)). Furthermore, 3-ketoacido-CoA-acyl carrier protein reductase (Taguchi K. et al., FEMS Microbiology

It is also useful to use Letters 176: 183-190 (1999).

 As long as these genes have substantial enzymatic activity, mutations such as deletions, substitutions, and insertions may occur in the nucleotide sequences of the genes. However, in the case of a heterologous gene, there is no guarantee that it will be translated into a protein having a function in Candida maltosa as described above, so it is desirable to change the amino acid codon.

 More preferably, both the polyester synthase gene and the acetoacetyl-CoA reductase gene can be used.

[0082] In the present invention, a phaC derived from Aeromonas serrata (Japanese Unexamined Patent Publication No. 10-108682, Fukui

 T. et al., FEMS Microbiology Letters, 170: 69-75 (1999)) and phbB (GenBank: J04987) derived from Ralstonia'eutropha.

[0083] A gene encoding phaC derived from Aeromonas' rabies was designed to be expressed in Candida's maltosa, and was prepared to replace asparagine at the 149th position from the amino terminal in the amino acid sequence with serine. The nucleotide sequence of the obtained DNA (phaCacl49NS) is shown in SEQ ID NO: 2.

 The nucleotide sequence of a DNA designed to express the gene encoding phbB derived from Ralstonia eutropha in Candida maltosa is shown in SEQ ID NO: 3.

[0084] However, the nucleotide sequence shown in these SEQ ID NOs is not limited thereto, and any nucleotide sequence may be used as long as the amino acid sequence of the enzyme is a nucleotide sequence expressed in Candida maltosa. Can be used. More preferably, a gene encoding “(serine Z alanine Z cystine) — (lysine Z arginine Z histidine) bite isine”, which also has three amino acid sequences as peroxisome orientation signals at the carboxyl terminus of these genes involved in polyester synthesis. Can be used. Here, for example, (serine Z alanine Z cysteine) means serine, alanine or cysteine. V, which means that it is misaligned (WO03Z033707).

 [0085] The gene expression cassette in yeast is obtained by ligating a DNA sequence such as a promoter, 5, an upstream activating sequence (UAS), etc., to the 5 'side of the gene, and a poly A It is prepared by ligating DNA sequences such as additional signal and terminator. Any of these DNA sequences can be used as long as they function in the yeast.

 [0086] The promoter and terminator used can be any sequence as long as it functions in yeast. Promoters include those that express constitutively and those that express inductively, and any promoter may be used. In the present invention, the promoter and terminator preferably function in Candida maltosa, and the promoter and terminator are more preferably derived from Candida maltosa. More preferably, it is a promoter having strong activity in the carbon source used.

 For example, when oils and fats are used as the carbon source, the promoters include the ALK1 gene (GenBank: D00481) promoter ALKlp (WOO 1Z88144) and the ALK2 gene (GenBank: X55881) promoter ALK2p of Candida maltosa. Can be. In addition, a promoter (Kogure, Abstracts of 2002 Annual Meeting of the Japanese Society of Agricultural Chemistry, pl91) with improved promoter activity by adding multiple ARR (alkane Responsible Region) sequences upstream of these promoters Number 4) can also be used. As the terminator, terminator ALKlt (WO01Z88144) of the ALK1 gene of Candida maltosa can be used. The base sequence of the promoter and Z or the terminator may be a base sequence in which one or more bases are deleted, substituted and Z or added, as long as the sequence functions in Canzaida maltosa. Is also good.

 The promoter is 5 ′ upstream of a gene encoding an enzyme involved in the synthesis of a polyester to which a DNA encoding a peroxisome orientation signal is added, and the terminator is a polyester having a DNA encoding a peroxisome orientation signal. Each is linked to the 3 'downstream of the gene encoding the enzyme involved in the synthesis.

[0087] The promoter and terminator are linked to the structural gene, and the gene expression cassette of the present invention is ligated. The method of constructing the kit is not particularly limited. In addition to the present example, a PCR method can also be used to prepare a restriction enzyme site. For example, the method described in WO01Z88144 can be used.

 [0088] In order to integrate this expression cassette into the yeast chromosome in a site-specific manner, a gene fragment having a sequence homologous to the chromosomal gene to be introduced is placed at both ends of the DNA in which the expression cassette and the gene serving as the selection marker are linked. Can be used. As a selectable marker, the ADE1 gene or the like that can be naturally deleted by intramolecular homologous recombination described in (1) above can also be used. The number of expression cassettes in the DNA for introduction is not limited, and shoes may be used if they can be produced.

 [0089] As a place for inserting the target gene, the sequence of the gene has been elucidated! It can be used at any location as long as it is located. Even if the gene sequence is unknown, it is possible to analyze the gene sequence based on the chromosomal gene sequence of a closely related yeast of which the gene sequence is known, so it can be inserted into virtually all gene sites It is. As a method for analyzing the gene sequence, it is possible to obtain from the yeast chromosome DNA library to be introduced using the Saccharomyces cerevisiae whose sequence has been analyzed or a homologous gene fragment of Candida albicans as a probe using the hybridization method. it can. Probes can be prepared using PCR or the like. The chromosomal DNA library can be prepared by a method known to those skilled in the art.

 [0090] As an example, the HIS5 gene is inserted as a marker gene into the URA3 disruption DNA-1 used for the URA3 gene disruption described in (1), and is involved in polyester synthesis between the URA3 gene fragment site and the HIS5 gene. By inserting a gene expression cassette, DNA can be prepared that specifically inserts the target gene into the disrupted URA3 site on the yeast chromosome using histidine requirement as a marker. As a method for introducing a gene, the method for electrotransfer described in (1) can be used.

[0091] The strain of the present invention can be transformed using a plurality of selectable markers to produce various strains into which a plurality of gene expression cassettes have been introduced. If the ADE1 gene, etc., which can be naturally deleted by intramolecular homologous recombination, is used, it is possible to restore the selectable marker, so that gene transfer can be performed at many places. In addition, a plasmid capable of autonomous replication in yeast can be used together. [0092] Polyester production by culturing yeast transformed with a gene expression cassette involved in polyester synthesis can be performed as follows.

 As the carbon source used for the culture, any carbon source can be used as long as yeast can assimilate. When the expression of the promoter is inducible, an inducer may be added at an appropriate time. Inducers may be the primary carbon source. As a nutrient other than the carbon source, a medium containing a nitrogen source, inorganic salts, and other organic nutrients can be used. The culture temperature may be any temperature at which the bacteria can grow, but is preferably 20 ° C to 40 ° C. The culture time is not particularly limited, but may be about 17 days. Thereafter, the polyester may be recovered from the obtained cultured cells or culture.

 [0093] In a preferred embodiment of the present invention, fats and oils, fatty acids, alcohols, n-alkanes and the like can be used as the carbon source. Examples of oils and fats include rapeseed oil, coconut oil, palm oil, palm kernel oil and the like. Fatty acids include saturated and unsaturated fatty acids such as butanoic acid, hexanoic acid, octanoic acid, decanoic acid, lauric acid, oleic acid, palmitic acid, linoleic acid, linolenic acid, and myristic acid, and esters and salts of these fatty acids. And the like. These can be mixed and used. In addition, in the case of V that cannot be assimilated or oil or fat that cannot be efficiently assimilated, it can be improved by adding lipase to the medium. Furthermore, by transforming the lipase gene, fat-and-oil assimilation ability can be imparted.

 [0094] Examples of the nitrogen source include ammonia, ammonium chloride, ammonium sulfate, ammonium phosphate, and other ammonium salts, as well as peptone, meat extract, yeast extract, and the like. Be

Examples of the inorganic salts include potassium phosphate monobasic, potassium phosphate dibasic, magnesium phosphate, magnesium sulfate, sodium salt, and the like.

 Other organic nutrients include, for example, amino acids such as glycine, alanine, serine, threonine, and proline; vitamins such as vitamin B1, vitamin B12, biotin, nicotinamide, pantothenic acid, and vitamin C. Can be

 Examples of the inducer include glucose and galactose.

[0095] A number of methods have been reported for the recovery of bacterial cell strength. In the present invention, For example, the following method can be used. After completion of the culture, the cells are separated and recovered from the culture solution using a centrifuge or the like, and the cells are washed with distilled water, methanol and the like, and then dried. At this stage, a step of disrupting the cells may be added. Polyester is extracted from the dried cells using an organic solvent such as chloroform. Cell components are removed from the organic solvent solution containing the polyester by filtration or the like, and a poor solvent such as methanol or hexane is added to the filtrate to precipitate the polyester. Then, the supernatant is removed by filtration or centrifugation, and the precipitated polyester is dried to recover the polyester.

 [0096] The obtained polyester can be analyzed by, for example, gas chromatography, nuclear magnetic resonance, or the like.

 [0097] Next, the novel yeast transformant of the second invention will be described.

 The second present invention is a yeast transformant into which a polyhydroxyalkanoic acid synthase gene and an acetoacetyl CoA reducing enzyme gene have been introduced, wherein two or more of these genes have been introduced. It is a yeast transformant characterized by having. (I) Host

 As the yeast referred to in the second aspect of the present invention, a yeast or the like deposited with a depository of a strain (for example, IFO, ATCC, etc.) that can introduce and transform a plurality of genes can be used. Preferably, Candida (Candida), Clavispora (Clavispora), Taliptococcus (Cryptococcus), and Denoriomyces (Denoliomyces) in terms of resistance to hydrophobic substances such as linear hydrocarbons. Genus Debaryomyces, genus Roderomyces, genus Pichia, genus Rhodotorula, genus Sporidiobolus, genus Stephanoascus, genus Yarr owia ) Can be used.

 Among these yeasts, the genus Candida is particularly preferred in that the analysis of the chromosomal gene sequence has been advanced, a host-vector system can be used, and the ability to assimilate linear hydrocarbons and oils and fats is high.

[0098] Among the genus Candida, it is preferable to use the species exemplified in the first aspect of the present invention, particularly in view of high assimilation ability of linear hydrocarbons and fats and oils. Among these species, the growth rate and infectivity are particularly preferred, especially the maltosa species.

 [0099] In the production of the yeast transformant of the present invention, if a selectable marker gene having properties such as drug resistance and nutritional requirement is also introduced into the host at the time of the transformation, the selection after transformation can be achieved. Only transformed strains can be selected by utilizing drug resistance and auxotrophy due to the expression of the marker gene.

 As a selectable marker gene, a gene that imparts resistance to cycloheximide-G418, hygromycin B, or the like can be used. In addition, a gene that complements auxotrophy can be used as a selection marker. These may be used alone or in combination.

[0100] However, for example, when a gene that complements auxotrophy is used as a selectable marker gene, an auxotroph that functions similarly to the selectable marker gene and whose host gene does not function substantially does not function. A sex-disrupting strain is required. Such auxotrophic disrupted strains (mutants) are not limited to those in which the target mutations can be obtained by random mutagenesis using a mutagen such as nitrosoguanidine or ethyl methanesulfonic acid. In the present invention, the host prepared by the gene disruption method by homologous recombination described in the first present invention is considered to have a high possibility of being contained, and as a result may be affected by a growth rate or the like. It is preferable to use.

 In the case of performing the transformation a plurality of times, the type of the marker according to the number of times of the transformation is required. Alternatively, as described below, it is necessary to restore the selection marker by removing the selection marker gene after introducing the gene-introducing DNA containing the selection marker gene into the host. In the examples of the present invention, a multiple auxotrophic gene-disrupted strain was used. Here, the DNA for gene transfer can cause homologous recombination with a gene on a chromosome in a microbial cell. Shows the DNA into which the target gene can be inserted.

[0101] The Candida maltosa AHU-71 strain, which is a ADE1, HIS5 and URA3 gene disruption strain, used in the examples of the present invention, was produced in the first present invention, and is Candida 'maltosa. It was prepared by the method described in Example 3 below using 16 strains of AC. In particular, when performing multiple transformations in the above-described host, if the yeast can use a plurality of selectable marker genes from the beginning without disrupting genes such as auxotrophy, the yeast transformant of the present invention can be efficiently used. Can be made.

 [0102] (II) PHA synthase gene and acetoacetyl CoA reductase gene

 The PHA synthase gene is not particularly limited, but a synthase gene for synthesizing a polyester obtained by copolymerizing 3-hydroxyalkanoic acid represented by the above general formula (1) is preferable, and It is a synthase gene for a copolymerized polyester P (3HB-CO-3HH) obtained by copolymerizing the 3-hydroxybutyric acid represented by the formula and the 3-hydroxyhexanoic acid represented by the formula (3). Like,.

[0103] As the PHA synthase gene, for example, the PHA synthase gene described in JP-A-10-108682 can be used.

 In the second invention, an acetoacetyl CoA reductase gene is used together with the PHA synthase gene. As the acetoacetyl-CoA reductase gene, any enzyme gene that has the activity of reducing acetoacetyl-CoA and synthesizing (R) -3-hydroxybutyryl-CoA can be used. For example, Ralstonia-eutropha (GenBank: A AA21973), Pseudomonas sp. 61-3 (GenBank: T44361), Zoogloea Lamigera (GenBank: P23238), and enzyme genes derived from Alkali Genes latus SH-69 (GenBank: AAB65780) can be used.

Further, in addition to the above genes, other genes involved in PHA synthesis can be used. Other genes involved in PHA synthesis include, for example, the (R) -body-specific enolyl CoA hydratase (Fukui T) that converts enolyl CoA, an intermediate in the j8 oxidation pathway, to (R) -3-hydroxyacyl-CoA. Et al., FEMS Microbiology Letters, 170: 69-75 (1999), JP-A-10-108682), and j8 ketothiolase (Peoples OP, etc.) for dimerizing acetyl-CoA to synthesize 3-hydroxybutylyl-CoA. J. Biol. Chem. 264: 15298-15303 (1989)), 3-ketoacyl-CoA acyl carrier protein reductase gene (Taguchi K. et al., FEMS Microbiology Letters, 176: 183-190 (1999)). In particular, an enzyme gene having activity for synthesizing (R) -3-hydroxyhexanoyl-CoA is preferable. The present invention uses a PHA synthase gene (phaC) and an acetoacetyl-CoA reductase gene (phbB) simultaneously.

 In the present invention, phaC derived from Aeromonas' cabies (JP-A-10-108682, Fukui T., et al., FEMS Microbiology Letters, 170: 69-75 (1999)) and phbB (GenBank: J04987) can be used.Preferable as the phaC is one encoding an enzyme or a mutant derived from Aeromonas radiata having the amino acid sequence represented by SEQ ID NO: 5, and the above phbB is preferably SEQ ID NO: 6. Those encoding an enzyme or a mutant derived from Ralstonia eutropha consisting of the amino acid sequence represented by are preferred.

 As long as these genes have substantial enzymatic activity, mutations such as deletions, substitutions, and insertions may occur in the nucleotide sequences of the genes. However, in the case of a heterologous gene, there is no guarantee that it will be efficiently translated into a protein having a function in the host yeast. Therefore, it is desirable to optimize the codon of amino acid.

[0105] As described above in the first present invention, when a heterologous gene is expressed using Candida's maltosa as a host, it is preferable to convert only leucine codon (CUG) in principle, and more efficient expression Other amino acid codons may be matched to those of Candida's Maltosa to achieve this.

 [0106] The PHA synthase gene can be prepared and used by modifying the amino acid sequence to obtain a mutant having improved properties such as enzyme activity, substrate specificity, and thermal stability. A variety of useful mutation methods are known in the art. In particular, molecular evolution engineering techniques (Japanese Patent Application Laid-Open No. 2002-199890) are highly useful because a desired mutant can be quickly obtained. Using these techniques, several synthase mutants have been found in the past, and it has been confirmed that the activity of Escherichia coli is higher than that of the wild-type enzyme (T. Kichise et al., Appl. Environ. Microbiolol). 68, 2411-2419 (2002), Amara A. A. et al. Appl. Microbiol. Biotechnol. 59, 477-482 (2002)).

[0107] Further, it is also possible to identify a useful amino acid mutation on the basis of a three-dimensional structure of an enzyme gene or a predicted three-dimensional structure on a computer by using, for example, a program Shrike (Japanese Patent Application Laid-Open No. 2001-184831). It is possible. As phaC in the present invention, for example, PHA synthase obtained by performing at least one of the following (a)-(h) amino acid substitutions obtained by using these methods on the amino acid sequence of a PHA synthase gene derived from Monas caviae Those encoding the mutant can be used.

 (a) Asn—149 to Ser

 (b) Asp—171 Gly

 (c) Phe—246 to Ser or Gin

 (d) Tyr-318 ^ Ala

 (e) Ile—320 for Ser, Ala or Val

 (f) Leu—350 to Val

 (g) Phe—353 to Thr, Ser or His

 (h) Phe—lie 518

 [0108] Here, for example, "Asn-149" means the 149th asparagine in the amino acid sequence of SEQ ID NO: 5, and the amino acid substitution in (a) converts the 149th asparagine into serine. Means

 [0109] Examples of phaC and phbB include those of SEQ ID NOs: 2 and 3 exemplified in the first invention. [0109] The amino acid sequence of the enzyme gene is expressed in Candida 'maltosa. If it is a nucleotide sequence, V, such a nucleotide sequence can also be used.

 [0110] When phaC and phbB are expressed in cytosolic substrates (cytosol, cytosol), these genes can be used as they are by modifying these genes into genes that are localized in peroxinorm (WO03 / 033707).

 As a method for localizing phaC and phbB to peroxinorm, the method described in the first present invention can be used.

 [0111] In addition, the nine amino acid sequences “(arginine Z lysine) (leucine Z valine Z isoleucine) (5 amino acids) (histidine Z glutamine) (leucine Z alanine)” present near the N-terminus are also used as luxosome orientation signals. Known! / By inserting and adding DNAs encoding these sequences to genes involved in PHA synthesis, the enzyme genes can also be localized in the peroxinin.

[0112] Furthermore, these genes were mitochondrial so that phaC and phbB would be expressed in mitochondria. It can also be used after being modified into a gene to be orientated. In order to localize these genes to mitochondria, a protein expressed and localized in mitochondria may be bound to the amino terminal. Examples include cytochrome oxidase and TCA cycle-related enzymes. For example, a gene encoding at least 15 residues, preferably at least 40 residues from the amino terminal of the protein expressed in mitochondria is shifted in frame to the 5 'upstream of the gene involved in PHA synthesis. What is the use of the linked genes? At this time, a linker sequence may be inserted between the added fusion gene and the gene involved in PHA synthesis to avoid unnecessary collision of amino acid residues. The gene used for the fusion gene is preferably derived from the host yeast used for the transformation of the present invention, but is not particularly limited.

 [0113] These genes designed to be oriented to the cytosol, peroxynome, and mitochondria can be used alone or in combination of two or more. As phaC and phbB, those with a peroxisome orientation signal added are preferred! /.

 [0114] (III) Gene expression cassette

 The expression cassette for the PHA synthase gene and the acetoacetyl CoA reductase gene used in the present invention is obtained by ligating a DNA sequence such as a promoter and a 5 ′ upstream activation sequence (UAS) at the 5 ′ side upstream of the gene. It can be prepared by connecting a DNA sequence such as a polyA addition signal and a terminator to the 3 ′ downstream of the gene.

 In the present invention, it is preferable that a promoter and a terminator that function in yeast are connected to the PHA synthase gene and the acetoacetyl CoA reductase gene.

[0115] The promoter and terminator to be used may be any sequence as long as it functions in yeast. Promoters include those that express constitutively and those that express inductively, and any promoter may be used. As the above-mentioned promoter, a promoter having a strong and active carbon source used for culturing transformants is preferable. For example

When oil or fat is used as a carbon source, the promoter described in the first invention can be used as the promoter.

[0116] As the terminator, the terminator ALKlt (WO01Z88144) of the ALK1 gene of Candida maltosa can be used. The above promoter and The base sequence of Z or one of the terminators may be a base sequence in which one or more bases are deleted, substituted and Z or added, as long as they are sequences that function in the host used. In the present invention, the promoter and the terminator preferably function in Candida and more preferably function in Candida maltosa. More preferably, it is derived from Da'maltosa.

 [0117] In a preferred embodiment of the present invention, the promoter is a PHA synthase gene to which a DNA encoding a peroxisome orientation signal has been added, and an acetoacetyl CoA reductase to which a DNA encoding a peroxisome orientation signal has been added. The terminator is linked to the 5 'upstream of the gene, the terminator is a PHA synthase gene to which DNA encoding a peroxisome orientation signal is added, and the acetoacetyl CoA to which DNA encoding a peroxisome orientation signal is added. It is linked to the downstream of the reductase gene at 3, respectively (WO03Z033707).

 [0118] The method for linking the promoter and terminator to phaC and phbB to construct the gene expression cassette of the present invention is not particularly limited, and the same method as in the first present invention can be used.

 (IV) Transformant

 The number of introduced expression cassettes per yeast cell in the desirable form of the present invention is strong under a carbon source such as hydrocarbons and fatty acids, even when the ARR promoter used is used. Although the gene expression was induced, one copy was not sufficient, and the present invention showed that either the number of the expression cassettes for phaC or the number of expression cassettes for phbB required at least two copies. Unless the amount of substrate supplied for host PHA synthesis is rate-limiting, the number of expression cassettes to be introduced is preferably as large as possible. The preferred number of expression cassettes depends on the type of promoter used.When promoter ARRp is used, it is preferable to introduce two or more copies at the same time, and it is more preferable to introduce three or more copies at the same time.

This expression cassette can be inserted into a vector capable of autonomous replication in yeast and introduced into host yeast. It can also be inserted into the host yeast chromosome. Both introduction methods They can be used simultaneously.

 [0120] When the vector is introduced into a host yeast, for example, pUTUl (M. Ohkuma, et al.) Capable of autonomous replication in Candida 'maltosa

 al j. Biol. Chem., vol. 273, 3948—3953 (1998)) and the like.

[0121] When a method of inserting an expression cassette into a chromosome is used, for example, homologous recombination can be used. Among homologous recombination, the gene replacement method is preferred because a transgenic strain that does not return spontaneously can be obtained. To insert the expression cassette into the chromosome by the gene replacement method, first, the expression cassette and the gene to be the selection marker are linked, and then the expression cassette and the gene to be the selection marker are linked to both ends of the DNA to be introduced. DNA (gene transfer DNA) to which a gene fragment having a sequence homologous to the above-mentioned gene has been linked can be used.

 [0122] The site on the chromosome into which the expression cassette or the like is inserted is not particularly limited as long as it has no irreversible effect on the host. The length of the region of homology to the gene on the chromosome to be transferred, which is bonded to both ends of the DNA for gene transfer, is preferably at least 10 bases, more preferably at least 200 bases, and still more preferably at least 300 bases. Further, the homology of each end is preferably 90% or more, more preferably 95% or more. That is, at the site where the gene sequence is analyzed, the gene can be used as it is, or even if the gene sequence is unknown, the chromosomal gene sequence of a closely related yeast having a known gene sequence is used. This comes out.

 [0123] When the homology is low and homologous recombination is unlikely to occur, the gene at the introduced site on the chromosome can be cloned and used. In order to clone the gene at the transfer site on the chromosome, the entire sequence of the chromosomal gene is analyzed and PCR primers are designed based on the sequences of Saccharomyces cerevisiae and Candida albicans to increase the gene. You just have to do the width. Further, similarly to the first present invention, a yeast chromosome DNA library to be introduced can also be used.

The number of expression cassettes in the DNA for gene transfer is not limited and may be any number as long as it can be produced. [0124] As the selection marker gene in the DNA for gene transfer, a gene that complements auxotrophy as described above can be used as the selection marker gene. Further, a gene imparting resistance such as cycloheximide G418 or hygromycin B can also be used. These selectable marker genes can be used in a form that can be naturally deleted by intramolecular homologous recombination described later. In this case, since the selectable marker gene can be recovered, the DNA for gene transfer using the same selectable marker gene can be introduced many times, and the transformation can be easily performed.

 [0125] DNA for gene transfer for introducing these expression cassettes into a yeast host can be prepared by a method known to those skilled in the art using a plasmid or the like that grows autonomously in E. coli or the like. As an example, the HIS5 gene is inserted as a selectable marker gene into the URA3 disruption DNA-1 described in Example 1 described later, and a phaC expression cassette and a phbB expression cassette are inserted between the URA3 gene fragment site. When inserted, histidine requirement can be used as a marker to produce a gene transfer DNA that specifically inserts the target gene into the URA3 site on the yeast chromosome.

 [0126] A plasmid containing the DNA for gene transfer can be prepared in the same manner as in the first present invention. This plasmid can be used directly for yeast transformation.However, a homologous portion including the chromosome transfer region is cut out from a purified vector with an appropriate restriction enzyme, and then used as DNA for gene transfer. Desired ヽ. It is also possible to amplify using PCR method.

 [0127] Examples of the yeast transformation method include the method exemplified in the first present invention, and the electric pulse method is preferable in the present invention. Prepare competent cells from the host strain, and after electropulsing together with the DNA for gene transfer, do not include the selectable marker gene! / ヽ Transformants do not proliferate! ヽ Culture in medium and screen for strains in which the DNA for gene transfer has been inserted into the desired chromosomal site from the appearing colonies.

 Screening of the target gene-introduced strain can also be performed in the same manner as in the first present invention.

[0128] The transformant of the present invention can be produced by introducing the phaC expression cassette and the phbB expression cassette until the target expression cassette number is reached using the above method. [0129] Instead of using the multiple auxotrophic gene-disrupted strain used in the examples of the present invention, a wild-type strain or a strain having no auxotrophy and having no auxotrophy is involved in the PHA synthesis of the present invention. A yeast transformant into which the gene has been introduced multiple times can be obtained. For example, when introducing DNA for gene transfer using a drug resistance marker gene as a selectable marker gene, the concentration of the drug used for selecting a transformant may be increased each time the DNA for gene transfer is transformed. . Further, if the selective gene introduced into the chromosome after the single transformation is removed, it can be used again as a marker for gene introduction, and a large number of DNAs for gene introduction can be introduced. For this method, for example, a gene disruption method described in JP-A-2002-209574 can be used. Furthermore, by inserting hisG gene fragments at both ends of the selectable marker gene, the DNA for gene transfer is prepared so that the marker gene inserted by intramolecular homologous recombination can be removed after gene transfer. (Alani et al. Genetics, 116: 541-545 (1987)).

 [0130] One of the polyester producing strains obtained by the present invention, CM313—X2B strain (Accession number: FE RM BP-08622) was patented on February 13, 2004 by the National Institute of Advanced Industrial Science and Technology (AIST). It has been internationally deposited at the Biological Depository Center under the Budapest Treaty.

 [0131] (V) Recovery method of selection marker

 The method for recovering a selectable marker of the present invention is characterized in that the ADE1 gene is removed by performing intramolecular homologous recombination with Candida 'maltosa having the ADE1 gene as a selectable marker gene. When further transformation is performed by removing the ADE1 gene, the ADE1 gene can be used again as a selectable marker gene.

Until now, it has been known to remove the selectable marker gene by intramolecular homologous recombination in Saccharomyces cerevisiae etc.The method of removing the selectable marker gene by intramolecular homologous recombination in Candida maltosa is as follows. Unknown power. As described above, a drug resistance gene or a gene that complements auxotrophy can be used as the selectable marker gene. Examples of the selectable marker gene include the ADE1 gene, the URA3 gene, the HIS5 gene, and the like. Gene removal is color selectable Use the appropriate ADE1 gene.

In the method of the present invention, the ADE1 gene can be removed by intramolecular homologous recombination even when a gene homologous to the ADE1 gene is bound, but a portion of the ADE1 gene is located upstream or downstream of the ADE1 gene. It is preferable in that it is easier to produce and does not leave extra genes on the yeast chromosome.

There is no particular limitation on the gene fragment used for intramolecular homologous recombination of the selectable marker gene. A gene fragment in which the selectable marker gene does not substantially function may be used. In the examples of the present invention, a gene fragment at the 5 'end of the ADE1 gene was used, but a gene fragment at the 3' end can also be used. The marker single gene fragment linked to the selectable marker gene preferably has at least 10 bases, more preferably at least 200 bases, and even more preferably at least 300 bases. That is, a marker gene fragment may be inserted at the 5 'end or the 3' end of the selectable marker gene in the DNA for gene transfer described in (IV) above! In the method of the present invention, the ADE1 gene preferably has a base sequence represented by SEQ ID NO: 7. The nucleotide sequence represented by SEQ ID NO: 7 is derived from Candida maltosa. This method can be applied to marker genes other than the ADE1 gene.

Fig. 4 shows a model of marker recovery by intramolecular homologous recombination. In FIG. 4, the numbers in parentheses indicate the numbers from the 5 ′ end of the sequence registered in GenBank of the ADE1 gene!

Strains from which the inserted selectable marker gene has been removed by intramolecular homologous recombination can be concentrated and selected by various methods. For example, a nystatin enrichment method can be used. The cells cultured in an appropriate medium are inoculated into a minimal medium or the like and cultured. After washing the bacteria and culturing in a minimal medium without a nitrogen source, cultivate briefly in a minimal medium with a nitrogen source. By directly adding nystatin to this culture solution and aerobically culturing at 30 ° C for 1 hour, the strain having the marker gene can be preferentially killed. Spread this bacterial solution on an appropriate agar plate and incubate at 30 ° C for about 2 days. If the selectable marker gene to be removed is the ADE1 gene that requires adenine, the precursor substance accumulates when the ADE1 gene is disrupted and the yeast stains red. Can be If the marker gene is the URA3 gene, コ ロ Select a colony that grows in a medium in the presence of racil and 5-FOA (5-Fluoro-Orotic-Acid). If there is no such selection method, a replica method can be used.

 (VI) Method for Controlling Physical Properties of Polyester

 Further, the method of controlling the molecular weight of the polyester of the present invention is characterized in that, in the production of a polyester using a yeast transformant, the number of acetoacetyl-CoA reductase genes in the yeast transformant is controlled. It is.

 Further, the method for controlling the hydroxyalkanoic acid composition of the polyester of the present invention controls the number of polyhydroxyalkanoic acid synthase genes in the yeast transformant in the production of the polyester using the yeast transformant. It is characterized by the following.

That is, the composition and molecular weight of the hydroxyalkanoic acid of the polyester which is the target product of the present invention can be controlled by adjusting the expression levels of phaC and phbB. When the phaC expression cassette and the phbB expression cassette using the same promoter are used, in order to increase the composition of hydroxyhexanoic acid, the number of phaC expression cassettes must be This can be done by increasing the number. In addition, the molecular weight can be increased by increasing the number of introduced phbB expression cassettes relative to the number of introduced phaC expression cassettes.

 A transformant having such characteristics can be prepared by the method described in (IV) above. Further, even when the number of introduced expression cassettes is the same, control of the hydroxyalkanoic acid composition and molecular weight can be achieved by changing the strength of the promoter used.

(VII) Culture purification

 The method for producing a polyester of the present invention is characterized in that the polyester is collected from a culture obtained by culturing the above yeast transformant.

 The culturing of the yeast transformed with the PHA synthase gene and the expression cassette of phbB can be carried out in the same manner as the method for culturing the transformed yeast described in the first aspect of the present invention.

 [0136] A number of methods have been reported for recovering the cell virulence of polyester. For example, the method described in the first present invention can be used.

[0137] Analysis of the obtained polyester is performed by, for example, gas chromatography or nuclear magnetic resonance. More can be done. The GPC method can be used for measuring the weight average molecular weight. For example, after the recovered dried polymer is dissolved in chloroform, the solution can be analyzed using a GPC system manufactured by Shimadzu Corporation equipped with Shodex K805L (manufactured by Showa Denko KK) using the chromate form as a mobile phase. A commercially available standard polystyrene or the like can be used as the molecular weight standard sample.

 The invention's effect

 [0138] The yeast having a plurality of markers produced by gene disruption of the present invention can be expected to be used as a gene recombination host for highly efficient gene expression and production of gene expression products. Furthermore, it is also possible to add a marker by gene disruption, which leads to the development of a better host. Further, according to the present invention, it has become possible to efficiently produce a copolymerized polyester obtained by copolymerizing 3-hydroxyalkanoic acid, which has biodegradability and excellent physical properties, in yeast. Furthermore, it became possible to control the physical properties of the copolymerized polyester. In addition, it has become possible to efficiently perform gene transfer multiple times in yeast.

 BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described more specifically with reference to Examples. However, the present invention does not limit the technical scope to these embodiments.

 Reagents used for culturing yeast were commercially available from Wako Pure Chemicals unless otherwise noted.

 Further, in the practice of the present invention, many commercially available kits were used, and the procedures were carried out according to the attached instructions unless otherwise specified.

[0140] (Medium composition)

 LB medium: 10 g / L tryptone, 5 g / L yeast extract, 5 g / L salt. In case of LB plate, add agar to 16gZL.

 YPD medium: lOgZL yeast extract, 20gZL polypeptone, 20gZL glucose. In the case of a YPD plate, agar is kneaded to 20 gZL. For adenine-containing YPD medium, add 0.1 lgZL of adenine.

YM medium: 3gZL yeast extract, 3gZL malt extract, 5gZL bactopeptone, lOgZL group Lucas.

 SD medium: 6.7 g / L amino acid-free yeast-trogen base (YNB), 20 g / L glucose. In the case of adenine-containing medium, add 24 mg / L of adenine. In the case of ゥ -lysine-containing medium, add 0.1 lg / L of L-lysine. In the case of a histidine-containing medium, add 5 OmgZL of histidine. In case of SD plate, add agar to 20gZL.

 [0141] M medium: 0.5 g ZL magnesium sulfate, 0.5 lg ZL salt, 0.4 mg ZL thiamine, 0.4 mg ZL pyridoxine, 0.4 mg ZL calcium pantothenate, 2 mg ZL inositol, 0.002 mg ZL biotin, 0.05 mg ZL salt glide Iron, 0.07 mg ZL zinc sulfate, 0.1 OlmgZL boric acid, 0.1 OlmgZL copper sulfate, 0.1 OlmgZL potassium iodide, 87.5 mgZL potassium dihydrogen phosphate, 12.5 mg ZL monopotassium hydrogen phosphate, 0.1 lgZL Calcium chloride, 20g ZL glucose. In the case of M medium containing ammonium sulfate, lg / L ammonium sulfate is added. In the case of M medium containing ammonium sulfate and adenine, add lgZL ammonium sulfate and 24 mg / L adenine to the M medium. In the case of M medium containing ammonium sulfate and adenine peridine, add lgZL ammonium sulfate, 24 mg ZL adenine, and 0.1 lgZL peridine to the M medium. In the case of M medium containing ammonium sulfate and adenine histidine, add lgZL ammonium sulfate, 24 mg ZL adenine, and 50 mg ZL histidine to the M medium. In the case of M medium containing ammonium sulfate and adenine 'peridine' histidine, add lgZL of ammonium sulfate, 24 mgZL of adenine, 0.1 lgZL of lysine, and 50 mgZL of histidine to the M medium.

 [0142] M2 medium (12.75 g ZL ammonium sulfate, 1.56 g ZL potassium dihydrogen phosphate, 0.33 g ZL potassium monohydrogen phosphate trihydrate, 0.08 g ZL potassium dihydrogen salt, 0.5 g ZL Shiojiri sodium, 0.41 gZL magnesium sulfate · 7 hydrate, 0.4 gZL calcium nitrate · 7 hydrate, 0. OlgZL Shiridani (ΠΙ) · tetrahydrate), 2 wZv% palm oil And trace elements dissolved in hydrochloric acid (lgZmL iron sulfate (Π) · heptahydrate, 8 gZmL zinc sulfate (Π) · heptahydrate, 6.4 gZmL manganese sulfate (Π) · tetrahydrate, 0.1 g 8gZmL Copper sulfate (Π) · pentahydrate) Add 0.45ml / L. Add 20 g ZL of fats and oils as a carbon source.

[0143] Liquid culture of yeast was performed using a 50 ml test tube, a 500 ml Sakaguchi flask, a 2 L Sakaguchi flask, or a mini jar. For 50ml test tube 300rpm, 500ml Sakaguchi flask 100-110 rpm, 2 L Sakaguchi flask Shaking culture at 90-100 rpm. The culture temperature is 30 ° C for both liquid culture and plate culture.

 [0144] (Restriction enzyme treatment)

 The restriction enzyme treatment was performed under the reaction conditions recommended by the manufacturer, or in Molecular Cloning: A Laboratory Manual ^ Second Edition ^ old spring Harbor Laboratory Press (1989), edited by Sambrook et al.

 Example 1 Preparation of Complementary Vector, Disruption Gene and Marker-Recovery Disruption Gene [0145] pUTU-1, which is a vector for Candida maltosa having the URA3 gene as a marker and provided by the University of Tokyo as a marker, A vector in which the Sail restriction enzyme site in the URA3 gene has been disrupted by the Stratagene quick change kit using the primers (del-sal-5, del-sal-3) described in SEQ ID NOs: 8 and 9 pUTU-delsal Was prepared. From this pUT U-delsal, the URA3 gene was cut out with Sail and Xhol, and the cut out fragment was again introduced into the Xhol site of pUT U-1 to prepare a plasmid pUTU-2. The ADE1 gene on pUTA-1 (described in WO01 / 88144) was cloned into the Xhol site of pUTU-2 to prepare pUTU-2-Ade. The HIS5 gene cloned into pUC119 was cloned into the Sail site of the multicloning site of pUTU-1 to produce pUTUl-His. Further, the HIS5 gene was cloned at the Sail site of the multicloning site of pUTU-2-Ade to produce pUTU-2-Ade-His.

 [0146] The pUC19 multicloning site was changed to Notl-Sphl-Sail-Xhol-Nhel-Swal-EcoRI, and the pUC-Nx Sphl-Sall site was replaced with the 5 'end of the URA3 gene, about 350 bases. Using the primers (ura-sph-5, ura-sal-3) described in SEQ ID NOS: 10 and 11, plasmid pUTU-delsaU was amplified and cloned. PCR amplification of about 460 bases at the 3 'end of the URA3 gene at the Nhel-Swal site of this vector using the primers (ura-nhe-5, ura-swa-3) described in SEQ ID NOs: 12 and 13. Cloning. Thus, a plasmid containing DNA-1 for URA3 disruption was prepared.

[0147] Next, the full length of the A DEI gene of pUTA-1 was placed in the Sail-Xhol site of the plasmid containing DNA-1 for URA3 disruption, using the primers (ade-sal-5, ade- x Amplification using ho-3) to produce a plasmid containing DNA-2 for URA3 disruption Made.

 [0148] The plasmid containing DNA-3 for URA3 disruption (marker recovery type) was prepared by substituting approximately 630 bases at the 5 'end of the ADE1 gene with the primers (ade-xho-5, ade-nhe The DNA was amplified by PCR using -3) and cloned into the Xhol-Nhel site of the plasmid containing DNA-2 for URA3 disruption to complete it.

 [0149] The plasmid containing DNA for HIS5 disruption (marker recovery type) was prepared using the primer (his-sph-5) of SEQ ID NOS: 18 and 19 containing about 500 bases at the 5 'end of the HIS5 gene cloned into pUC119. Using the primers (his-nhe-5, his-swa-3) described in SEQ ID NOs: 20 and 21, URA3 was disrupted from the 3, terminal portion of the HIS5 gene using the primers (his-nhe-5, his-swa-3). The plasmids were prepared by sequentially closing each of the plasmids containing DNA-3 for Sphl-Sail site and Nhel-Swal site.

 (Example 2) URA3 gene disruption of AC16 strain

AC 16 strain was cultured overnight in YPD medium in a 10-ml large test tube. The pre-cultured yeast was inoculated into YM medium so as to form a 1 ml ZlOOml Sakaguchi flask, and after culturing for 6 hours, the cells were collected. The bacteria were suspended in 20 ml of 1 M Sorbitol and washed three times. Finally, the cells were suspended in 0.5 ml of 1 M Sorbitol to obtain a competent cell. To 0.1 ml of the competent cells, 0.1 mg of DNA obtained by treating a plasmid containing DNA-2 for disrupting URA3 of Example 1 with Sphl and Swal, and adding 0.1 mg of the DNA, was subjected to gene transfer by an electric pulse method. After applying electric noise, 1 ml of 1 M Sorbitol was added to the cuvette, left for 1 hour under ice-cooling, and sown on an SD plate. Chromosome DNA was extracted from the resulting colonies using a chromosome extraction kit Gentle-kun (Takara Shuzo). Each 5 μg of the obtained chromosomal DNA was digested with restriction enzymes using three methods, Scal, EcoT14I, ScaI + EcoT14I, cut, and electrophoresed on a 0.8% agarose gel. Molecular cloning: 1 Laboratory transfer from Genore to Hybond N + filter (Amersham) according to A Laboratory Manual ^ second Edition 9.31-9.57, Cold Spring Harbor Laboratory Press (1989). The probe for Southern blot detection used was a Seal-Ndel fragment (340 bp), which is a sequence in the URA3 gene, which was enzymatically labeled with a Genelmage Labeling 'detection kit (Amersham). After washing, washing was performed, and DNA bands were detected with the fluorescent coloring reagent of the same kit. Detection van When the wild type strain was compared with that of the wild type strain IAM12247, the wild-type strain showed a band of about 570 bp.The DNA treated with Seal + EcoT14I also showed a new 1840 bp band indicating the insertion of the ADE1 gene into the URA3 gene. Selected. This strain showed theoretical values even in bands treated with Seal or EcoT14I, confirming that one of the URA3 genes was correctly destroyed by the ADE1 gene.

 Next, 0.5 mg of DNA obtained by amplifying a plasmid containing DNA-2 for URA3 disruption by PCR with the primers of SEQ ID NOS: 10 and 13 was added to the competent cells prepared from this strain in the same manner as described above. Then, gene transfer was performed by the electric nosling method in the same manner as described above. After applying the pulse, the cells were inoculated into 100 ml of YM medium and cultured for 1 晚. The cells were collected and cultured for 1 hour in M medium (without ammonium sulfate). After recovering the cells, the cells were transferred to M medium (with ammonium sulfate), cultured for 6 hours, nystatin was added to a final concentration of 0.1 OlmgZml, and the cells were further cultured for 1 hour. The cells were washed and spread on an SD plate containing adenine. Genomic DNA was extracted from the red colonies that appeared. The genome of the adenine-requiring strain obtained here was amplified using primers ura3-5 and ura3-3 described in SEQ ID NOs: 22 and 23, and the 0.9 kbp of the URA3 gene that was intact in the original strain was obtained. 2.3 kbp of DNA was lost, and 0.7 kbp of DNA was amplified instead. When amplification was performed using the primers adeYlO-5 and adeY1670-3 described in SEQ ID NOS: 24 and 25, 1. Only Okbp DNA was amplified. From these facts, it was determined that the entire region of the ADE1 gene used for site-specific URA3 gene disruption was specifically removed. One of the adenine-requiring strains was named U-1 strain and used in the following experiments.

Next, using the clone obtained using the U-1 strain, a competent cell was prepared. 0.1 ml of this combined cell, and a plasmid containing DNA-3 for URA3 disruption treated with restriction enzymes SphI and Swal, were added with 0.04 mg of purified DNA, and gene transfer was performed by the electric pulse method. The cells were spread on an SD plate containing Perysin and incubated at 30 ° C. The emerged colonies were replicated on an SD plate and an SD plate containing Perysin, and a Periracil-requiring strain was selected. From this, chromosomal DNA was recovered and subjected to PCR amplification using the primers ura3-5 and ura3-3 described in SEQ ID NOs: 22 and 23, and the 0.9 kbp band of the intact URA3 gene disappeared. DNA for URA3 destruction—3 rhinoceros 2.9 kbp was amplified. When amplification was performed using primers adeYl10-5 and adeY1670-3 described in SEQ ID NOS: 24 and 25, 1.5 kbp of the ADE1 gene, which was not amplified in the original strain, was amplified. Based on these facts, it was determined that the disruption gene 3 was introduced specifically for the URA3 gene, all the URA3 genes were disrupted, and the requirement for peracil was obtained. The auxotrophy of the obtained strain was confirmed to be complemented by the vector pUTU-2. This ゥ rasil-requiring strain was named Candida maltosa U-35.

 Next, Candida's maltosa U-35 strain was cultured overnight in 10 ml of YPD medium. After harvesting, the cells were cultured in M medium (without ammonium sulfate) for 1). After recovery of the cells, the cells were transferred to a peridine-containing M medium (with ammonium sulfate), cultured for 7 hours, and nystatin was added to a final concentration of 0.1 Olmg / ml, followed by further 1 hour of culture. After washing the cells, the cells were cultured in an M medium containing adenine and peridine (with ammonium sulfate) for 1 hour. Collect the cells, culture them in M medium (without ammonium sulfate), transfer them to M medium containing peridine (with ammonium sulfate), culture them for 7 hours, and treat them with nystatin as before. And spread it on an SD plate containing adenine and peridine. After culturing, the obtained red colonies were replicated on an SD plate, an SD plate containing peridine, and an SD plate containing adenine and peridine, and confirmed to be a clone showing both auxotrophy for adenine and peracil. Genomic DNA was extracted from this strain and subjected to PCR amplification using the primers ura 3-5 and ura 3-3 shown in SEQ ID NOs: 22 and 23. As a result, the ADE1 gene was introduced into the URA3 gene. Band disappeared, and instead, 1.2 kbp, the size of the ADE1 gene fragment remaining in the URA3 gene, was amplified. When amplification was performed using the primers adeYl10-5 and adeY1670-3 described in SEQ ID NOs: 24 and 25, the parent strain was amplified by the ADE1 gene 1.5 kbp. 1. Only the Okbp band, which is the size of the ADE1-disrupted gene, was observed. These results indicate that the ADE1 gene and the ADE1 gene fragment in the URA3 gene naturally lost the ADE1 gene by homologous recombination within the same gene, and that the gene marker was easily recovered. . This double auxotroph of Aden-Peracil was named Candida maltosa UA-354.

 (Example 3) HIS5 gene disruption in AC16 strain and marker recovery method

Prepare competent cells from the U-1 strain prepared in Example 2 and contain HIS5 disruption DNA The plasmid was treated with the restriction enzymes Sphl and Swal, and purified DNA04 (0.4 mg) was used for gene transfer by the electric pulse method. The conditions are the same as in Example 2. The cells were spread on a histidine-containing SD plate and incubated at 30 ° C. Genomic DNA was recovered from the colonies that appeared. The primers for the flanking site of the homology with the HIS 5 gene of the disrupting gene in the HIS 5 gene, that is, the primers for the HIS 5 gene not included in the disrupting gene, his-sal2 and his- 1900 ( When genomic DNA was amplified using SEQ ID NOs: 26 and 27), a strain was selected that amplifies 3.4 kbp, the size of HIS5 disruption DNA, along with an intact HIS5 gene size of 1.9 kbp band. .

 [0155] Using this strain, nystatin was concentrated in the same manner as described in Example 2. Spread on an adenine-containing SD plate, genomic DNA was extracted from the resulting red colonies, and genomic DNA was amplified using primers his-sal2 and his-1900 (SEQ ID NOs: 26 and 27). Amplification of only the 1.9 kbp band, which is the size of the HIS5 gene, was observed, and 3.4 kbp, the size containing the disrupting gene, was not amplified. When PCR amplification was performed using the primers ade-xho-5 and his-swa-3 (SEQ ID NOs: 16 and 21), a 1.2 kbp band in which the ADE1 gene fragment was bound to the HIS5 gene was amplified. From this, it was confirmed that the obtained adenine-requiring strain was obtained as a result of intramolecular homologous recombination that could not be achieved with Rivalant.

[0156] Next, a competent cell was prepared from the obtained adenine-requiring strain, and a plasmid containing DNA for disrupting HIS5 was treated with the restriction enzymes Sphl and Swal, and purified DNA05. Gene transfer was performed. The cells were spread on an SD plate containing histidine and incubated at 30 ° C. The emerging colony was replicated on an SD plate and an SD plate containing histidine to obtain a histidine-requiring strain. Genomic DNA was extracted from the obtained histidine-auxotrophic strain, and genomic DNA was amplified using primers his-sal2 and his-1900 (SEQ ID NOs: 26 and 27). A strain that amplifies 3.4 kbp, a size containing a disrupting gene in addition to the 1.9 kbp band, which is the size of 5 genes, was selected. In this strain, the ADE1 gene was integrated into the HIS5 gene by PCR using primers his-sal2 and ade-xho-3 (SEQ ID NOs: 26 and 15). I confirmed that. This histidine-requiring strain was named Candida maltosa CH-I strain.

 [0157] Using the CH-I strain, nystatin was concentrated in the same manner as described in Example 2.

 It was spread on an SD plate containing adenine and histidine, and genomic DNA was extracted from the obtained red colonies. When genomic DNA was amplified using the primers his-sal2 and his-1900 (SEQ ID NOs: 26 and 27), the 1.9 kbp band, the size of the parental disrupted HIS5 gene in all strains, was obtained. Only 3.4 kbp, the size containing the disrupting gene, was not amplified. Histidine and adenine auxotrophy were confirmed by replicating to an SD plate, a histidine-containing SD plate, and an adenine and histidine-containing SD plate, and a double auxotroph of adenine 'histidine was completed. One of them was named Candida maltosa AH-15 strain.

 [0158] Next, a competent cell was prepared from the AH-15 strain, a plasmid containing DNA-3 for URA3 disruption treatment was treated with the restriction enzymes Sphl and Swal, and purified DNA was collected at 0.025 mg, followed by an electric pulse method. Was introduced. The cells were spread on an SD plate containing peridine and histidine, and incubated at 30 ° C for 2 days. The emerging colonies were replicated on SD plates containing histidine and SD plates containing peridine and histidine, and strains requiring periracil were selected. From this, chromosomal DNA was recovered and subjected to PCR amplification using primers ura3-5 and ura3-3 (SEQ ID NOs: 22 and 23). As a result, the 0.9 kbp band of the intact URA3 gene disappeared and was instead destroyed. Amplification of 2.9 kbp, which is the size of the gene for use, was confirmed. One of the histidine 'Duracil dual auxotrophs was designated Candida' Maltosa HU-591.

[0159] Using the HU-591 strain, nystatin was concentrated in the same manner as described in Example 2. The cells were spread on an SD plate containing adenine, histidine and peridine, and genomic DNA was extracted from the obtained red colonies. PCR amplification was performed using primers ura3-5 and ura3-3 (SEQ ID NOs: 22 and 23). The ADE1 gene was introduced into the URA3 gene. The 2.9 kbp band disappeared, and the ADE1 gene fragment was replaced with the URA3 gene. A strain in which 1.2 kbp was amplified was selected. The requirements for perylase, histidine, and adenine are as follows: SD plate containing adenine, histidine and peridine, S plate containing histidine and peridine Confirmed by replicating to D plate, SD plate containing adenine and peridine, SD plate containing adenine and histidine, SD plate containing adenine, SD plate containing histidine, SD plate containing peridine, and SD plate, and adenine 'histidine' A triple auxotroph was completed. This strain was named Candida maltosa AHU-71.

 [0160] When the marker-recovery-type disruption gene is used, the frequency of occurrence of adenine-requiring strains is similar to that when adenine-destroying DNA is used, but the time required to obtain the target strain is about half. Could be shortened. Furthermore, it was easy to analyze without having to consider insertion into the target external site when introducing the disrupted gene.

 [0161] As shown in this example, it was shown that marker genes can be easily recovered using intramolecular homologous recombination.

 (Example 4) Confirmation of the ability to utilize fats and oils

 Using the finally completed triple auxotroph AHU-71 strain, jar culture was performed to confirm that there was no problem with growth when using oils and fats as a carbon source. The AHU-71 strain was transformed with the plasmid pUTU2-Ade-His, and colonies were formed on SD plates. A control obtained by transforming the plasmid pUTA-1 into the AC16 strain was used. Seeds were prepared by culturing in a Sakaguchi flask using 150 ml of SD medium. The jar culture was performed by charging 1.8 L of the M2 medium into a 3 L jar arm amenter manufactured by Malvisi. The temperature was 32 ° C., the number of agitation was 500 rpm, and the aeration rate was lwm. Palm kernel oil was fed as a carbon source at 1.9 mlZh up to 11 hours from the start of cultivation, 3.8 mlZh up to 24 hours, and 5.7 mlZh thereafter. Over time, 10 ml of the culture was sampled, washed with methanol, dried, and the amount of dry cells was measured. As shown in FIG. 2, the AHU-71 strain showed the same growth as the AC 16 strain. From this, it was confirmed that this strain was able to disrupt the gene without impairing the ability to utilize fats and oils.

 (Example 5) Construction of expression cassette for enzyme gene involved in synthesis of polyester

In order to express polyester synthase in Candida maltosa, a promoter derived from Candida maltosa was connected upstream, and a terminator was connected downstream, respectively. ALK2 gene (GenBank: X55881) promoter ARRp, a promoter with an ARR sequence added upstream, was ligated, and ALKlt, a terminator of the ALK1 gene of Candida maltosa (GenBank: D00481), was linked 3 ′ downstream. ARRp binds the EcoRI Xhol linker to the Pstl site of the gene (SEQ ID NO: 4) donated by the University of Tokyo, and binds the synthetic DNA shown in SEQ ID NO: 28 to the EcoT14I site. Was converted into a form that can be cut out with. After pUAL1 (WO01 / 88144) was cut with EcoRI, blunt ends were performed and ligation was performed to prepare pUAL2 from which the EcoRI cleavage site had been removed. pUAL2 was digested with PuvIlZPuvI and bound to the SmalZPuvII site of pSTV28 (Takara Shuzo) to produce pSTALl. This pSTALl was digested with EcoRlZNdel and ligated with ARRp as described above to produce pSTARR.

 [0164] A peroxisome orientation signal was added to the carboxy terminus so that the phaCacl49NS of SEQ ID NO: 2 was oriented to the peroxinorme. Ser-Lys-Leu (SKL) amino acid at the carboxy terminus was used as the peroxisome orientation signal after the addition. Next, phaCacl49NS that had been closed to pUCNT was transformed into type III, and the gene was amplified using the primers of SEQ ID NOs: 29 and 30 and ligated to the Ndel and Pstl sites of pSTARR to construct pSTARR-phaCacl49NS. The nucleotide sequence was confirmed using the primers of SEQ ID NOs: 31 to 35. The nucleotide sequence was determined using a DNA sequencer 310 Genetic Analyzer manufactured by PERIKIN ELMER APPLIED BIOSYSTEMS.

Next, the carboxy terminus of the acetoacetyl CoA reductase gene (phbB) derived from Ralstonia eutropha II H16 strain (ATCC17699) derived from Ralstonia eutropha H16 (ATCC17699) described in SEQ ID NO: 3 in which codons were converted for chemically synthesized Candida maltosa Was added by amplifying a peroxisome orientation signal with the primers of SEQ ID NOs: 36 and 37, and then binding to the Ndel and Pstl sites of pSTARR described above to construct pSTARR-phbB. The nucleotide sequence was confirmed by the same method as described above.

[0166] Two synthetic enzyme expression cassettes cut out from pSTARR-pha Cacl49NS with Sail and Xhol were introduced into the Sail site of pUTA-1 which is a vector for Candida's maltosa, to prepare pARR-149NSx2. Further, one phbB expression cassette cut out from pSTARR-phbB with Sail and Xhol was introduced into the Sail site of this vector to prepare pARR-149NSx2-phbB.

[0167] DNA for introducing a heterologous gene into the disrupted HIS5 gene portion on the chromosome of Candida maltosa was prepared using the DNA for disrupting HIS5 described in Example 1 . The HIS5 disruption DNA was cut with Sail and Xhol to remove the ADE1 gene, and instead a plasmid was prepared in which pUTU-delsal was introduced with the URA3 gene cut with Sail and Xhol. An expression cassette was cut out from the above pSTARR-phaCacl49NS with Sail and Xhoi at the Sail site of this vector, and ligated. Next, the expression cassette was excised from pS TARR-phaCacl49NS with Sail and Xhol at the Xhol site of this plasmid and ligated to prepare a plasmid containing DNA-1 for introduction.

 Further, a phbB expression cassette cut out from pSTARR-phbB with Sail and Xhol was bound to the Xhol site of the plasmid containing the DNA-1 for introduction to prepare a plasmid containing the DNA-2 for introduction.

 [0168] DNA for introduction of a heterologous gene into the disrupted URA3 gene portion on the chromosome of Candida maltosa was used for the introduction of a plasmid containing DNA-1 for URA3 disruption described in Example 1 Produced. A plasmid was prepared in which the HIS5 gene amplified by PCR with the primers of SEQ ID NOs: 38 and 39 was introduced into the Sail-XhoI site of the plasmid containing DNA-1 for URA3 disruption. An expression cassette was cut out from the above pSTARR-pha Cacl49NS with Sail and Xhol at the Sail site of this plasmid and ligated. Next, an expression cassette was excised from pSTARR-phbB at the Xhol site of this vector using Sail and Xhol and ligated to prepare a plasmid containing DNA-3 for introduction. At this Sail site, instead of the phaC expression cassette, a plasmid containing an introductory DNA-4 linked with a phbB expression cassette was also prepared.

A schematic diagram of the prepared DNA for gene transfer 1-4 is shown in FIG. In FIG. 3, the numbers in parentheses indicate the numbers from the 5 ′ end of the gene registered in GenBank of the used gene fragment. ADE1 gene: D00855, URA3 gene: D12720, HIS5 gene: XI 7310. The thick frame indicates a homologous site with chromosomal DNA. (Example 6) Construction of recombinant strain

 Using the Candida maltosa AHU-71 strain prepared in Example 3, the competent cells for electrotransduction were prepared by the method described in Example 2. To the competent cells, 0.05 mg of DNA-1 and 2 for introduction treated with restriction enzymes Notl and Swal were electrotransformed and spread on an SD plate containing adenine and histidine. Chromosomal DNA was prepared from the colonies that appeared, and PCR was performed using the primers represented by SEQ ID NOS: 26 and 27. In addition to the 1.9 kbp gene corresponding to the disrupted HIS5 gene, a colony in which a gene having a size corresponding to DNA-1 and 2 for introduction was amplified was selected as a strain introduced into the HIS5 gene site. Furthermore, PCR using various primers confirmed that the introduced genes had no deletions.

 Next, from these strains, similarly, competent cells for electrotransfer were prepared. 0.05 mg of the transfection DNA-3 and 4 treated with the restriction enzymes Notl and Swal were electrotransformed into the resulting combinant cells and spread on an adenine-containing SD plate. Chromosomal DNA was prepared from the colonies that appeared, and PCR was performed using the primers represented by SEQ ID NOS: 22 and 23. Of the 0.7 kbp and 1.2 kbp genes corresponding to the disrupted URA3 gene, amplification of either gene was not observed, and instead, genes having sizes equivalent to the transfer DNA-3 and 4 were amplified. Colonies were selected as strains that were introduced at the URA3 gene site. Furthermore, it was confirmed by PCR using various primers that there was no deletion in these introduced genes.

[0170] The Candida 'maltosa AC16 strain was transformed with the pARR-149NSx2-phbB prepared in Example 5, and the strain into which two copies of the phaCacl49NS expression cassette and one copy of the phbB expression cassette were inserted was used as strain A. . A strain prepared by inserting DNA-1 and 4 into the Candida maltosa AHU-71 strain and having two copies of the phaCacl49NS expression cassette and two copies of the phbB expression cassette inserted on the chromosome was designated as strain B. A strain prepared by using DNA-1 and 3 for introduction into Candida's maltosa AHU-71 strain and having 3 copies of the phaCacl49NS expression set and 1 copy of the phbB expression cassette inserted on the chromosome was designated as strain C. A strain in which 3 copies of the phaCacl 49NS expression cassette and 2 copies of the phbB expression cassette were inserted into the chromosome of Candida maltosa AHU-71 strain using DNA-2 and 3 for transfer was designated as strain D. It was. The C strain was transformed with pARR-149NSx2-phbB to prepare an E strain into which 5 copies of the phaCacl49NS expression cassette and 2 copies of the phbB expression cassette were introduced. The D strain was transformed with pARR-14 9NSx2-phbB to prepare an F strain in which 5 copies of the phaCacl49NS expression cassette and 3 copies of the phbB expression cassette were introduced. Transformation of pARR-149NSx2-phbB into a strain in which two copies of the phaCacl49NS expression cassette and three copies of the phbB expression cassette have been inserted into the chromosome prepared using the transfer DNAs 2 and 4, and 4 copies of the phaCacl49NS expression cassette Then, a G strain was prepared in which 4 copies of the phbB expression cassette were introduced. This G strain was named CM313-X2B and deposited internationally (FERM BP-08622). Similarly, as a control, a strain in which pUTA-1 was transformed into 16 Candida's maltosa AC strain (control-1) and a strain in which pARR-149NSx2 was transformed (control-2) were also prepared. Table 1 summarizes the strains produced.

[0171] [Table 1]

(Example 7) Polymer production using recombinant strain

A recombinant Candida maltosa strain into which a gene required for polymer production was introduced was cultured as follows. As the medium, SD medium was used for pre-culture, and M2 medium containing palm kernel oil as a carbon source was used as a production medium. The glycerol stock 5001 of each recombinant strain was inoculated into a 500 ml Sakaguchi flask containing 50 ml of the preculture medium, cultured for 20 hours, and 10 vZv% was inoculated into a ² L Sakaguchi flask containing 300 ml of the production medium. This was cultured under the conditions of a culture temperature of 30 ° C, a shaking speed of 90 rpm, and culture for 2 days. The cells are collected from the culture by centrifugation, suspended in 80 ml of distilled water, and suspended in an ultrahigh-pressure homogenizer (APV, Rannie 2000, 1). (5000 Psi for 15 minutes), and then centrifuged. The obtained precipitate was washed with methanol and lyophilized. The obtained dried cells were pulverized and lg was weighed. To this, 100 ml of black form was added, and the mixture was stirred overnight to extract. The cells were removed by filtration, the filtrate was concentrated to 10 ml with an evaporator, and about 50 ml of hexane was added to the concentrate to precipitate a polymer, followed by drying. The composition of the obtained polymer was analyzed by NMR analysis (ClEOL, JNM-EX400). The weight-average molecular weight was measured by dissolving 10 mg of the recovered dried polymer in 5 ml of black-mouthed form, and then using this solution on a Shimadzu GPC system equipped with Shodex K805L (300x8 mm, 2 tubes connected) (Showa Denko KK). The form used was analyzed as the mobile phase. A commercially available standard polystyrene was used as a molecular weight standard sample. Table 2 shows the results.

[0173] [Table 2]

[0174] The above results clearly show that the use of the novel gene-disrupted yeast and the novel mutant of the present invention enables extremely efficient production of PHA. Also, the introduction of two or more copies of the gene involved in polyester biosynthesis is extremely important for efficient production of polyester, and the molecular weight and composition can be controlled by the number of expression cassettes introduced. It was clear that there was something.

 Industrial applicability

[0175] The yeast having a plurality of markers produced by gene disruption of the present invention It can be expected to be used as a recombinant host for highly efficient gene expression and production of gene expression products. Further, according to the present invention, it has become possible to efficiently produce a copolymerized polyester obtained by copolymerizing 3-hydroxyalkanoic acid having biodegradability and excellent physical properties in yeast. Furthermore, it is also possible to add a marker by gene disruption, which leads to the development of a better host.

According to the present invention, a copolymerized polyester obtained by copolymerizing 3-hydroxyalkanoic acid represented by the general formula (1) having biodegradability and excellent physical properties is efficiently produced in yeast. It became possible. In addition, it has become possible to efficiently perform gene transfer multiple times in yeast.

Brief Description of Drawings

FIG. 1 is a simple schematic diagram of a DNA for destruction prepared in an example.

FIG. 2 is a graph comparing the growth properties of novel gene-disrupted yeasts.

 FIG. 3 is a schematic diagram of gene-transfer DNA-14 prepared and used in Example 4.

FIG. 4 is a schematic diagram of intramolecular homologous recombination.

Claims

The scope of the claims

 [1] Peracil-requiring gene-disrupted yeast in which the URA3 gene of chromosomal DNA has been disrupted by homologous recombination with a URA3 DNA fragment.

[2] A histidine-requiring gene-disrupted yeast in which the HIS5 gene of chromosomal DNA has been disrupted by homologous recombination with a HIS5 DNA fragment.

[3] By homologous recombination with ADE1 DNA fragment and URA3 DNA fragment,

An adenine- and peracil-required gene-disrupted yeast in which the DEI gene and the URA3 gene have been disrupted.

 [4] A gene-disrupting yeast requiring adenine and histidine, in which the ADEI gene and the HIS5 gene of chromosome DNA have been disrupted by homologous recombination with the ADE1 DNA fragment and the HIS5 DNA fragment.

 [5] A gene-disrupted yeast requiring peracil and histidine, wherein the URA3 gene and the HIS5 gene of chromosomal DNA have been disrupted by homologous recombination with a URA3 DNA fragment and a HIS5 DNA fragment.

 [6] A gene-disrupting yeast requiring adenine, peracil, and histidine, wherein the ADE1 gene, URA3 gene, and HIS5 gene of chromosomal DNA have been disrupted by homologous recombination with the ADE1 DNA fragment, URA3 DNA fragment, and HIS5 DNA fragment.

 [7] Yeasts of the genus Candida, Clavisbora, Talyptococcus, Debaryomyces, Mouth Delomyces, Metoshnikouia, Pichia, Rhodosporidium, Rhodotonorella, Spolidiobolas, Stefano The gene-disrupted yeast according to any one of claims 16 to 17, which belongs to the genus Acasus or the genus Yarrowia.

 [8] The gene-disrupted yeast according to any one of [16] to [16], wherein the yeast is of the genus Candida.

[9] Yeast power Alcantans, ancudensis, atmosphaerica, azyma ia, bertae, olanku, butyri, conglobata, dendronemaia, ergastensi s, fluviatilis, iriearichii, gropengiesseii, cantita incommun is, insectrum, laureliae, maltosa, melibiosica, membranifaciens ia, mesenterica, natalensis, oregonensis, palmioleophila, parapsi losis, psudointermedia, quercitrusa, rhagu, rugosa, saitoana The gene-disrupted yeast according to any one of claims 16 to 17, which is any one of, sake, schatavu ^ a, sequanensis®, shehatae, sorbophila, tropicalis, valdiviana, and viswanathii.

[10] The gene-disrupted yeast according to any one of claims 16 to 16, wherein the yeast is Candida maltosa.

 [11] The URA3 gene-disrupted yeast according to claim 1, which is Candida's maltosa U-35 (FERM P-19435).

 [12] The HIS5 gene disrupting yeast according to claim 2, which is Candida's maltosa CH-I (FERM P-19434).

 [13] The AD according to claim 3, which is Candida 'Maltosa UA-354 (FERM P-19436).

El gene and URA3 gene disrupted yeast.

[14] The ADE1 gene and HIS5 gene-disrupted yeast according to claim 4, which is Candida maltosa AH-I5 (FERM P-19433).

[15] The UR according to claim 5, which is Candida 'Maltosa HU-591 (FERM P-19545).

A3 gene and HIS5 gene disrupted yeast.

[16] A according to claim 6, which is Candida's maltosa AHU-71 (FERM BP-10205).

DE 1 gene, URA3 gene and HIS 5 gene disrupted yeast.

[17] The transformant of the gene-disrupted yeast according to any one of [1] to [16], which is transformed with a DNA sequence containing a homologous or heterologous gene.

[18] A method for producing a gene expression product, comprising collecting a homologous or heterologous gene expression product from a culture obtained by culturing the transformant according to claim 17.

[19] The method for producing a gene expression product according to claim 18, wherein the gene expression product is a polyester.

 [20] A yeast transformant into which a polyhydroxyalkanoic acid synthase gene and an acetoacetyl CoA reductase gene have been introduced, wherein at least two or more of these genes have been introduced. A yeast transformant characterized by the following.

21. The yeast transformation according to claim 20, wherein a peroxisome orientation signal is added to the polyhydroxyalkanoate synthase gene and the acetoacetyl CoA reductase gene. body.

 22. The yeast transformant according to claim 20, wherein a promoter and a terminator that function in yeast are connected to the polyhydroxyalkanoate synthase gene and the acetoacetyl CoA reductase gene.

 [23] The yeast transformant according to any one of claims 20 to 22, wherein the yeast is of the genus Candida.

 [24] Yeast power Albicans, ancudensis, atmosphaerica, azyma ia, bertae, olanku, butyri, conglobata, dendronemaia, ergastensi s, fluviatilis, iriearichii, gropengiesseii, sp. , Incommun is, insectrum, laureliae, maltosa, melibiosica, membranifaciens, mesenterica, natalensis, oregonensis, palmioleophila, parapsi losis, psudointermedia, quercitrusa, rhagu, rugosa, saito , Sake ^ a, schatavu ^ a, sequanensis®, shehatae, but sorbophila species, tropicalis species, valdiviana species or viswanathii species, wherein the yeast transformant according to claim 20 to 22, wherein the shear force is 1. .

 [25] The yeast transformant according to any one of claims 20 to 22, wherein the yeast is Candida maltosa.

 [26] The polyhydroxyalkanoate synthase gene encodes an enzyme or a mutant derived from Aeromonas californiae having the amino acid sequence represented by SEQ ID NO: 5; Yeast transformant.

 [27] Genetic power of polyhydroxyalkanoic acid synthase derived from Aeromonas' rabies The following (a)-(h) V encodes a polyhydroxyalkanoic acid synthase mutant having at least one amino acid substitution 27. The yeast transformant according to claim 26, which is a yeast transformant.

 (a) Asn—149 to Ser

 (b) Asp—171 Gly

 (c) Phe—246 to Ser or Gin

 (d) Tyr-318 ^ Ala

(6) 361 116-320: Ala or Val (f) Leu—350 to Val

 (g) Phe—353 to Thr, Ser or His

 (h) Phe—518 lie

 [28] the yeast trait according to any one of claims 20 to 27, which encodes an enzyme or a mutant derived from Ralstonia eutropha having the amino acid sequence represented by SEQ ID NO: 6; Convertible.

 [29] The yeast trait according to [20], wherein the polyhydroxyalkanoic acid is a copolymer obtained by copolymerizing a 3-hydroxyalkanoic acid represented by the following general formula (1). Convertible.

 [Chemical 1]

 (In the formula, R represents an alkyl group having 113 carbon atoms.)

 [30] A polyhydroxyalkanoic acid is a copolymer polyester obtained by copolymerizing 3-hydroxybutyric acid represented by the following general formula (2) and 3-hydroxyhexanoic acid represented by the following general formula (3). 30. The yeast transformant according to claim 20, wherein the yeast transformant is a yeast transformant.

 [Formula 2]

CH ₃

HO—— CH ~ CH ₂ -C— OH (2)

 II

 o

[Formula 3]

C3H7

HO—— CH—— CH ₂ — C— OH (3)

 II

o

[31] A method for producing a polyester using the yeast transformant according to any one of claims 20 to 30, wherein the polyester is collected from a culture obtained by culturing the yeast transformant. A method for producing a polyester, comprising:

[32] A method for producing a polyester using the yeast transformant according to any one of claims 20 to 30.

A method for controlling the molecular weight of polyester by controlling the number of acetoacetyl-CoA reductase genes in a yeast transformant.

[33] In the production of a polyester using the yeast transformant according to any one of claims 20 to 30, by controlling the number of polyhydroxyalkanoate synthase genes in the yeast transformant. A method for controlling the hydroxyalkanoic acid composition of a polyester.

[34] A method of recovering the selection ability, which comprises removing the ADE1 gene by performing intramolecular homologous recombination with Candida maltosa having the ADE1 gene as a selection marker gene.

 [35] The method according to claim 3, wherein a part of the ADE1 gene is linked upstream or downstream of the ADE1 gene.

4. The method for recovering the selection marker according to 4.

[36] the ADE1 gene strength, or the base sequence strength represented by SEQ ID NO: 7;

35. The method for recovering a selection marker according to 35.